Devices, systems and methods for detecting viable infectious agents in a fluid sample

ABSTRACT

Various devices, systems and methods for detecting a susceptibility of an infectious agent to an anti-infective are described herein. A method comprises introducing a fluid sample to a first surface and a second surface; exposing the first surface to a first solution; exposing the second surface to a second solution, wherein the second surface comprises an anti-infective; sampling the first solution after exposing the first solution to the first surface; sampling the second solution after exposing the second solution to the second surface; monitoring a first electrical characteristic of a first sensor exposed to the first solution sampled; monitoring a second electrical characteristic of a second sensor exposed to the second solution sampled; and comparing the first electrical characteristic and the second electrical characteristic to assess the susceptibility of the infectious agent to the anti-infective.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/209,754 filed on Aug. 25, 2015, the content of whichis incorporated herein by reference in its entirety. This applicationalso incorporates by reference the content of U.S. patent applicationSer. No. 14/297,603 filed on Jun. 5, 2014 and U.S. patent applicationSer. No. 14/599,190 filed on Jan. 16, 2015 in their entireties.

FIELD OF THE INVENTION

The present disclosure relates generally to in vitro detection ofinfectious agents and, more specifically, to devices, systems, andmethods for detecting viable infectious agents in a fluid sample.

BACKGROUND

Infections caused by anti-infective resistant infectious agents ormicrobes are a significant problem for healthcare professionals inhospitals, nursing homes, and other healthcare environments. Forexample, such infections can lead to a potentially life-threateningcomplication known as sepsis where chemicals released into thebloodstream by an infectious agent can trigger a dangerous whole-bodyinflammatory response as well as a vasoactive response causing fever,low blood pressure, and possibly death. When faced with such aninfection, a preferred course of action is for a clinician to useanti-infective compounds judiciously, preferably only those necessary toalleviate the infection. However, what occurs most frequently today isthat until the organism is identified and tested for drug sensitivity,broad spectrum anti-infectives, often multiple drugs, are given to thepatient to insure adequacy of treatment. This tends to result inmultiple drug resistant infectious agents. Ideally, the sensitivity ofthe infectious agent would be detected soon after its presence isidentified. The present disclosure presents devices, systems, andmethods for accomplishing this goal.

Existing methods and instruments used to detect anti-infectiveresistance in infectious agents include costly and labor intensivemicrobial culturing techniques to isolate the infectious agent andinclude tests such as agar disk diffusion or broth microdilution whereanti-infectives are introduced as liquid suspensions, paper disks, ordried gradients on agar media. However, those methods require manualinterpretation by skilled personnel and are prone to technical orclinician error.

While automated inspection of such panels or media can reduce thelikelihood of clinician error, current instruments used to conduct theseinspections are often costly and require constant maintenance. Inaddition, current instruments often rely on an optical read-out of theinvestigated samples requiring bulky detection equipment and access topower supplies. Most importantly, these methods require days to obtain aresult, as the infectious agents must reproduce several times indifferent media prior to being exposed to the anti-infective todetermine their susceptibility.

In addition, such methods and instruments often cannot conduct suchtests directly on a patient's bodily fluids and require lengthy samplepreparation times.

As a result of the above limitations and restrictions, there is a needfor improved devices, systems, and methods to quickly and effectivelydetect anti-infective resistant infectious agents in a patient sample.

SUMMARY

Various devices, systems and methods for detecting the susceptibility ofan infectious agent in a patient sample to one or more anti-infectivesare described herein.

In one embodiment, a method for detecting the susceptibility of aninfectious agent to one or more anti-infectives can include introducinga fluid sample to a first surface and a second surface, exposing thefirst surface to a first solution, and exposing the second surface to asecond solution. The second surface can comprise an anti-infective.

In some instances, the fluid sample can comprise the infectious agentand the infectious agent can be introduced to the first surface or thesecond surface through the fluid sample. The method can also includedetermining the presence of the infectious agent in the fluid sample.

The method can include sampling the first solution after exposing thefirst solution to the first surface. The method can also includesampling the second solution after exposing the second solution to thesecond surface. The method can include monitoring a first electricalcharacteristic of a first sensor exposed to the first solution sampled.The method can include monitoring a second electrical characteristic ofa second sensor exposed to the second solution sampled.

The method can further include comparing the first electricalcharacteristic and the second electrical characteristic to assess thesusceptibility of the infectious agent to the anti-infective. Comparingthe first electrical characteristic and the second electricalcharacteristic can include determining a difference between the firstelectrical characteristic and the second electrical characteristic. Thedifference between the first electrical characteristic and the secondelectrical characteristic can be a result of a difference in a solutioncharacteristic of the first solution and the second solution. Thedifference in the solution characteristic of the first solution and thesecond solution can result from a difference in a molecular count, aconcentration of an ion, and/or a solution temperature.

The first surface can be a filter surface or a well surface. The secondsurface can be separate from the first surface and can be anotherinstance of the filter surface or the well surface. At least one of thefirst surface and the second surface can be a non-clogging filter. Inaddition, at least one of the first surface and the second surface cancomprise pores of sequentially smaller pore size.

The infectious agent can be, but is not limited to, a bacteria, afungus, a virus, or a prion. The first sensor and the second sensor canbe housed by a protective chamber and the protective chamber can be anelectrically isolated environment, a temperature controlled chamber,and/or a light controlled chamber. The first solution can be directed tothe first surface by a pump. The second solution can also be directed tothe second surface by a pump.

In another embodiment, a method for detecting a susceptibility of aninfectious agent to an anti-infective can include introducing a fluidsample to a first surface and a second surface, exposing the firstsurface to a first solution, and exposing the second surface to a secondsolution. The second surface can comprise an anti-infective.

In some instances, the fluid sample can comprise the infectious agentand the infectious agent can be introduced to the first surface or thesecond surface through the fluid sample. The method can also includedetermining the presence of the infectious agent in the fluid sample.

The method can include sampling the first solution after exposing thefirst solution to the first surface. The method can also includesampling the second solution after exposing the second solution to thesecond surface. The method can include monitoring a first electricalcharacteristic of a sensor exposed to the first solution sampled. Themethod can also include monitoring a second electrical characteristic ofthe sensor exposed to the second solution sampled.

The method can further include comparing the first electricalcharacteristic and the second electrical characteristic to assess thesusceptibility of the infectious agent to the anti-infective. Comparingthe first electrical characteristic and the second electricalcharacteristic can include determining a difference between the firstelectrical characteristic and the second electrical characteristic. Thedifference between the first electrical characteristic and the secondelectrical characteristic can be a result of a difference in a solutioncharacteristic of the first solution and the second solution. Thedifference in the solution characteristic of the first solution and thesecond solution can result from a difference in a molecular count, aconcentration of an ion, and/or a solution temperature.

The first surface can be a filter surface or a well surface. The secondsurface can be separate from the first surface and can be anotherinstance of the filter surface or the well surface. At least one of thefirst surface and the second surface can be a non-clogging filter. Inaddition, at least one of the first surface and the second surface cancomprise pores of sequentially smaller pore size.

The infectious agent can be, but is not limited to, a bacteria, afungus, a virus, or a prion. The first sensor and the second sensor canbe housed by a protective chamber and the protective chamber can be anelectrically isolated environment, a temperature controlled chamber,and/or a light controlled chamber. The first solution can be directed tothe first surface by a pump. The second solution can also be directed tothe second surface by a pump.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates one embodiment of a system for detecting asusceptibility of an infectious agent to one or more anti-infectives.

FIG. 2 illustrates another embodiment of the system for detecting asusceptibility of an infectious agent to one or more anti-infectives.

FIG. 3 illustrates another embodiment of the system for detecting asusceptibility of an infectious agent to one or more anti-infectives.

FIG. 4A illustrates a side view of an embodiment of a substrate havingan active sensor disposed on the substrate and an external reference.

FIG. 4B illustrates a side view of an embodiment of a substrate havingthe active sensor and an on-chip reference electrode disposed on thesubstrate.

FIG. 5A illustrates a side view of an embodiment of a substrate havingthe active sensor and a control sensor disposed on the substrate and anexternal reference electrode.

FIG. 5B illustrates a side view of an embodiment of a substrate havingthe active sensor, the control sensor, and the on-chip referenceelectrode disposed on the substrate.

FIG. 6A illustrates a side view of an embodiment of the active sensorand the control sensor each having an extended gate and an externalreference electrode.

FIG. 6B illustrates a side view of an embodiment of the active sensorand the control sensor each having an extended gate and an on-chipreference electrode.

FIG. 7 illustrates an embodiment of the system on a disposable strip.

FIG. 8 illustrates the analyzer and the reader processing signalsoutputted by the active sensor and the control sensor.

FIG. 9 illustrates experimental results of experiments conducted usingthe methods and systems described herein.

FIG. 10 illustrates additional experimental results of experimentsconducted using the methods and systems described herein.

FIG. 11 illustrates an embodiment of a method for detecting asusceptibility of an infectious agent to one or more anti-infectives.

FIG. 12 illustrates another embodiment of the method for detecting asusceptibility of an infectious agent to one or more anti-infectives.

FIG. 13 illustrates yet another embodiment of the method for detecting asusceptibility of an infectious agent to one or more anti-infectives.

FIG. 14 illustrates another embodiment of the method for detecting asusceptibility of an infectious agent to one or more anti-infectives.

FIG. 15 illustrates a further embodiment of the method for detecting asusceptibility of an infectious agent to one or more anti-infectives.

FIG. 16 illustrates another embodiment of the method for detecting asusceptibility of an infectious agent to one or more anti-infectives.

FIG. 17 illustrates another embodiment of a system for detecting asusceptibility of an infectious agent to one or more anti-infectives.

FIG. 18 illustrates another embodiment of a method for detecting asusceptibility of an infectious agent to one or more anti-infectives.

DETAILED DESCRIPTION

Variations of the devices, systems, and methods described herein arebest understood from the detailed description when read in conjunctionwith the accompanying drawings. It is emphasized that, according tocommon practice, the various features of the drawings may not be toscale. On the contrary, the dimensions of the various features may bearbitrarily expanded or reduced for clarity and not all features may bevisible or labeled in every drawing. The drawings are taken forillustrative purposes only and are not intended to define or limit thescope of the claims to that which is shown.

FIG. 1 illustrates an embodiment of a system 100 for detecting orassessing a susceptibility of an infectious agent 102 to ananti-infective 104. The system 100 can comprise a fluid delivery device106, a filter housing 108 containing a filter 110, a substrate 112, anda reader 114. The substrate 112 can have one or more sensors 116disposed on a surface of the substrate 112. The substrate 112 can becomprised of a polymeric material, a metal, a ceramic, a semiconductorlayer, an oxide layer, an insulator, or a combination thereof. Thesystem 100 can also include an analyzer 118. In the embodiment shown inFIG. 1, the analyzer 118 can be disposed on a surface of the substrate112. In other embodiments, the analyzer 118 can be a standalone unit ordevice coupled to the substrate 112.

The sensors 116 can include one or more active sensors 120, one or morecontrol sensors 122, or a combination thereof. As illustrated in theembodiment shown in FIG. 1, the one or more active sensors 120 andcontrol sensors 122 can be disposed on the same side surface of thesubstrate 112. In other embodiments not shown in FIG. 1, the activesensors 120 and the control sensors 122 can be disposed on differentsurfaces of the substrate 112, different substrates 112, or acombination thereof. For example, FIG. 1 shows the substrate 112 havingfour sensors 116; however, it is contemplated that the substrate 112 cancomprise any number of sensors 116. In one embodiment, at least one ofthe sensors 116 can be an ion-sensitive field effect transistor (ISFET).The sensors 116 will be discussed in more detail in the sections thatfollow.

The system 100 can detect or assess the level of susceptibility of theinfectious agent 102 to an anti-infective 104. In some instances, thefluid sample 124 can comprise the infectious agent 102. The fluid sample124 can include a bodily fluid such as blood, serum, plasma, urine,saliva, joint fluid, semen, wound material, spinal fluid, mucus, or acombination thereof. In other embodiments, the fluid sample 124 can alsoinclude an environmental fluid such as liquids sampled from a stream,river, lake, ocean, contamination site, quarantine zone, or emergencyarea. The fluid sample 124 can also be a food sample. The system 100 candetermine the presence of the infectious agent 102 in the fluid sample124 before detecting or assessing the level of susceptibility of theinfectious agent 102 to the anti-infective 104.

The infectious agent 102 can be any metabolizing single ormulti-cellular organism including a bacteria or fungus. The infectiousagent 102 can also be a virus or a prion.

In certain embodiments, the infectious agent 102 can be a bacteriaselected from the genera consisting of Acinetobacter, Aeromonas,Bacillus, Bacteroides, Citrobacter, Enterobacter, Escherichia,Klebsiella, Morganella, Pandoraea, Proteus, Providencia, Pseudomonas,Ralstonia, Raoultella, Salmonella, Serratia, Shewanella, Shigella,Stenotrophomonas, Streptomyces, Staphylococcus, Enterococcus,Clostridium or any combination thereof. In other embodiments, theinfectious agent 102 can be a fungus selected from the genera consistingof Candida, Cryptococcus, or any combination thereof. In anotherembodiment, the infectious agent 102 can include amoeba. In furtherembodiments, the infectious agent 102 can be cancer cells and theanti-infectives 104 can be chemotherapeutics or other cancer treatments.

As illustrated in FIG. 1, the fluid delivery device 106 can deliver orinject the fluid sample 124 into the filter housing 108 in step 1A. Thefluid sample 124 injected can comprise the infectious agent 102. In theexample embodiment shown in FIG. 1, the fluid delivery device 106 can bea pump. For example, the fluid delivery device 106 can be a syringepump, a pneumatic pump, or a hydraulic pump. In other embodiments notshown in FIG. 1, the fluid delivery device 106 can be an injectioncartridge, a microfluidic channel, a pipette, a reaction tube, acapillary, a test tube, a combination thereof, or a portion therein.

The filter housing 108 can be a container or vessel configured to secureor enclose the filter 110. The filter housing 108 can also be aprotective chamber. The protective chamber can be an electricallyisolated environment, a temperature controlled chamber, and/or a lightcontrolled chamber. For example, the filter housing 108 can be a housingof a syringe filter.

The filter 110 can be a non-clogging filter. The filter 110 can have anon-clogging filter surface. The filter 110 can also have filter poresof sequentially smaller pore size. For example, the filter 110 can havelarger filter pores at the top of the filter and progressively smallerfilters pores toward the bottom of the filter 110. Although not shown inFIG. 1, it is contemplated by this disclosure that the filter 110 canrefer to a plurality of filters in a stacked arrangement.

The filter 110 can be a mesh or matrix for isolating or separating theinfectious agent 102 or other molecules or cells from the supernatant ofthe fluid sample 124. In certain embodiments, the filter 110 can beselected from the group consisting of cellulose acetate, regeneratedcellulose, nylon, polystyrene, polyvinylidene fluoride (PVDF),polyethersulfone (PES), polytetrafluorethylene (PTFE), glass fiberpolypropylene, or a combination thereof.

The filter 110 can comprise a filter surface 126. The filter surface 126can be the portion of the filter 110 used to isolate or trap theinfectious agent 102. The filter surface 126 can include an externalsurface, an internal surface extending into the filter 110, or acombination thereof. The filter housing 108 can have at least oneopening 128 which allow fluid or supernatant from the fluid sample 124to evacuate the filter housing 108. For example, step 1A can include theadditional step of discarding the fluid or supernatant from the fluidsample 124 through the opening 128 after isolating the infectious agent102 on the filter surface 126.

In another embodiment not shown in FIG. 1, a stimulus solution can beadded to the fluid sample 124 before introducing the fluid sample 124 tothe filter 110. The stimulus solution can be a nutrient or growthsolution. The stimulus solution can have a different composition thanthe nutrient solution 130. The stimulus solution can be a super nutrientsolution.

In an alternative embodiment not shown in FIG. 1, the fluid sample 124can be pre-filtered in a step before step 1A. This pre-filtering stepcan involve filtering the fluid sample 124 using another instance of thefilter 110, a microfluidic filter, or a combination thereof to filterout other larger cellular components including blood cells or epithelialcells from the fluid sample 124 when the fluid sample 124 is composed ofbodily fluid.

The same fluid delivery device 106 or another fluid delivery device 106can also be used to deliver or inject a nutrient solution 130 to thefilter housing 108 in step 1B. The fluid delivery device 106 cancontinuously or periodically expose the filter surface 126 containingthe infectious agent 102 with the nutrient solution 130. In oneembodiment, the nutrient solution 130 can be composed of a buffercontaining bacto-tryptone, yeast extract, sodium chloride and anycombinations thereof. In another embodiment the nutrient solution caninclude a growth inducer. The growth inducer can be selected from thegroup consisting of a carbon-based inducer, a nitrogen-based inducer, amineral, a trace element, a biological growth factor, or any combinationthereof. For example, the growth inducer can include but is not limitedto glucose, ammonia, magnesium, or a combination thereof. For example,the nutrient solution 130 can be comprised of Tryptone, yeast extract,NaCl, and glucose.

The buffer in the nutrient solution 130 can be an acidic buffer or abasic buffer. The buffer can be used to counteract the buffering effectsof ions or substances present in the fluid sample 124 when the fluidsample 124 is composed of a bodily fluid.

The filter 110 comprising the infectious agent 102 can be heated to atemperature of between 30° C. and 40° C. and allowed to incubate for anincubation period 132 in step 1C. In one embodiment, the filter 110 canbe incubated while in the filter housing 108. In another embodiment, thefilter 110 can be removed from the filter housing 108 prior toincubation. In some embodiments, the filter 110 can be incubated withthe nutrient solution 130. The incubation period 132 can range from 15minutes to over one hour. In other embodiments, the incubation period132 can be less than 15 minutes. The incubation period 132 can beadjusted based on the type of infectious agent 102, such as the type ofbacteria, fungus, virus, or prion.

The incubation period 132 can also be adjusted based on the amount ofthe infectious agent 102 present in the fluid sample 124. For example,the incubation period 132 can be increased when the amount of theinfectious agent 102 is below a threshold amount. The filter 110 can beallowed to incubate with the nutrient solution 130 in order to promotethe proliferation of the infectious agent 102 on the filter surface 126.One advantage of incubating the filter 110 is to increase thesensitivity of the system 100 to small amounts of the infectious agent102. For example, incubating the filter 110 can allow the system 100 toreduce its level of detection. In one embodiment, the system 100 candetect as few as 500 bacteria per milliliter. In other embodiments, thesystem 100 can detect fewer than 500 bacteria per milliliter. In furtherembodiments, the system 100 can detect 10⁴ bacteria per milliliter.

After incubating the filter 110, the same fluid delivery device 106 oranother fluid delivery device 106 can then be used to expose the filtersurface 126 with additional nutrient solution 130 in step 1D. Oneadvantage of exposing the filter 110 with the additional nutrientsolution 130 is to prevent the filter housing 108, the filter 110, orthe environment housing the infectious agent 102 from becoming overlyacidified as a result of cellular activity, cellular metabolism, orgrowth undertaken by the infectious agent 102. For example, the filterhousing 108 or the filter 110 comprising the infectious agent 102 canbecome overly acidified as result of the infectious agent 102 undergoingcellular metabolism or growth.

As illustrated in the example embodiment shown in FIG. 1, the effluentor outflow from the exposure step of step 1D can be introduced orapplied to one or more of the sensors 116 disposed on the substrate 112.This effluent or outflow can be referred to as a sample effluent 134.

The sample effluent 134 can be introduced to one or more of the sensors116 disposed on the substrate 112 through the opening 128 in the filterhousing 108. The opening 128 can include a channel, a capillary, a tube,or a combination thereof. The sample effluent 134 can be sampled fromthe nutrient solution 130 exposed to the filter 110. The sample effluent134 can also be separated from the infectious agent 102 on the filtersurface 126 as the sample effluent 134 flows through the filter 110 onto the sensors 116. In these embodiments, the infectious agent 102 canbe kept separate or prevented from contacting any portion of the sensors116 disposed on the substrate 112.

The sample effluent 134 can comprise a solution characteristic 136. Thesolution characteristic 136 can refer to one or more attributes of thesolution making up the sample effluent 134. For example, the solutioncharacteristic 136 can include a concentration of a solute or anabsolute number of solutes in solution. The solution characteristic 136can include an amount or concentration of ions, organic molecules suchas amino acids, minerals, or other inorganic compounds in the sampleeffluent 134.

The solution characteristic 136 can vary as a result of ions, organicmolecules, or minerals produced by or attributed to the infectious agent102 on the filter surface 126. The solution characteristic 136 can be adirect or indirect byproduct of a cellular activity undertaken by theinfectious agent 102 such as cell metabolism or cell growth. In oneembodiment, the sample effluent 134 can comprise hydrogen ions (H⁺) as abyproduct of bacterial cell metabolism or growth. In other embodiments,the sample effluent 134 can comprise adenosine triphosphate (ATP),carbon dioxide (CO₂), lactic acid, carbonic acid, nitrates (NO₃ ⁻), or acombination thereof produced by or attributed to the infectious agent102.

After introducing the sample effluent 134 to the sensors 116, the samefluid delivery device 106 or another fluid delivery device 106 can beused to introduce an anti-infective 104 to the filter surface 126comprising the infectious agent 102 in step 1E. In the exampleembodiment shown in FIG. 1, the anti-infective 104 can be mixed withadditional nutrient solution 130 and the filter surface 126 comprisingthe infectious agent 102 can be exposed to additional nutrient solution130. In other embodiments, the anti-infective 104 can be introduced tothe filter surface 126 separate from the nutrient solution 130.

The anti-infective 104 can comprise a bacteriostatic anti-infective, abactericidal anti-infective, an anti-fungal anti-infective, an antiviralanti-infective, a prion inhibitor, or a combination thereof. In certainembodiments, the bacteriostatic anti-infective can comprise β-lactams,Aminoglycosides, Ansamycins Glycopeptides, Lipopeptides, Quinolones,Streptogramins, or any combination thereof. The bactericidalanti-infective can comprise Chloramphenicols, Macrolides,Oxazolidinones, Sulfonamides, Tetracyclines, any combination thereof, orfuture derivations thereof.

In another embodiment, the anti-infective 104 can be a bacterial growthinhibitor or stimulator. The bacterial growth inhibitor or stimulatorcan selectively inhibit or promote the growth of gram positive or gramnegative bacteria. The bacterial growth inhibitor or stimulator cancomprise a dye or a chemical compound or reagent. In some embodiments,the dye can include, but is not limited to, Methylene blue, Bromothymolblue, Eosin B, Safranin O, Crystal violet, or a combination thereof. Thechemical compound or reagent can include, but is not limited to, sodiumazide, bile salts, sodium chloride, tetrathionate, or a combinationthereof. The anti-infective 104 can also comprise a carbon source otherthan glucose, such as lactose, mannose, glycerol, or citrate to selectfor certain bacterial species. The bacterial growth inhibitor, thecarbon source, or a combination thereof can also be added to thenutrient solution 130.

In the example embodiment shown in FIG. 1, the filter 110 incubated instep 1C can be divided into two separate filters 110 with each filter110 having the infectious agent 102 on the filter surface 126. In thisembodiment, nutrient solution 130 containing the anti-infective 104 canbe exposed or introduced to one of the filters 110 comprising theinfectious agent 102 in step 1D and nutrient solution 130 without theanti-infective 104 can be exposed or introduced to the other filter 110in step 1E. In one embodiment, step 1D can occur concurrently or near intime with step 1E. In other embodiments, step 1D and step 1E can occursequentially.

In these embodiments, the sample effluent 134 resulting from theexposure step of step 1D can be introduced to a different sensor 116than the sensor 116 used to analyze the sample effluent 134 from step1E. Also, in these embodiments, the sensor 116 receiving the sampleeffluent 134 containing the anti-infective 104 can be referred to as theactive sensor 120 and the sensor 116 receiving the sample effluent 134without the anti-infective 104 can be referred to as the control sensor122.

In an alternative embodiment contemplated by the present disclosure, thesame filter 110 exposed to the nutrient solution 130 in step 1E can beexposed to a nutrient solution 130 containing the anti-infective 104 ata later point in time. In this embodiment, the sample effluent 134 fromthe exposure step comprising the anti-infective 104 can also beintroduced to the same sensor 116 as the sensor 116 used to measure thenon-anti-infective sample effluent 134 in step 1E.

In yet another embodiment contemplated but not shown in FIG. 1, portionsof the fluid sample 124 can be divided into multiple filter housings 108prior to step 1A. In this embodiment, each filter housing 108 cancontain a filter 110 comprising infectious agents 102 from the fluidsample 124 disposed on the filter surface 126. Each of the filterhousings 108 can be incubated and a variety of nutrient solutions 130,including nutrient solutions 130 lacking in anti-infective 104 orcontaining different types of anti-infectives 104, can be used to exposethe various filters 110. In this embodiment, the sample effluent 134from the various filter housings 108 can be introduced to differentsensors 116 on the substrate 112.

While FIG. 1 illustrates two of the four sensors 116 on the substrate112 being used to analyze sample effluent 134 from the fluid sample 124,it is contemplated that the substrate 112 can accommodate any number ofsensors 116 for receiving the sample effluent 134. For example, thesubstrate 112 can be a support or housing for a high throughput assayplate such as a 96 well plate, a 192 well plate, or a 384 well plate. Inthis example, each of the well plates can be in fluid communication withone or more sensors 116. In another embodiment, the sensors 116 can bepositioned directly underneath the filter housing 108.

The reader 114, the analyzer 118, or a combination thereof can beconfigured to monitor an electrical characteristic 800 (see FIG. 8) ofthe sensors 116 upon introducing the sample effluent 134 to the sensors116. For example, the reader 114 can monitor the electricalcharacteristic 800 of the sensors 116 by receiving one or more signalsfrom the analyzer 118 disposed on the substrate 112. In one embodiment,the analyzer 118 can comprise a controller to execute logical commandsconcerning the detection or comparison of the electrical characteristic800 of the sensors 116. In other embodiments, the controller can beintegrated with the reader 114 or another device coupled to the analyzer118.

The electrical characteristic 800 can include a current, a voltage, athreshold voltage, a capacitance, a resistance, a noise level, asubthreshold swing, a level of induction, or a combination thereofmeasured at or near the sensor 116. The reader 114 can be electricallyor communicatively coupled to the analyzer 118, the substrate 112, or acombination thereof to monitor the electrical characteristic 800 of thesensors 116 over time. The reader 114 can also be configured to providea read-out of the electrical characteristic 800 of the sensors 116.

In certain embodiments, the reader 114 can be a mobile device, ahandheld device, a tablet device, or a computing device such as a laptopor desktop computer. In other embodiments, the reader 114, the analyzer118, a combination thereof, or any portion therein can be integratedinto an ISFET probe or meter.

In the example embodiment shown in FIG. 1, the analyzer 118, the reader114, or a combination thereof can monitor the electrical characteristic800 of the sensors 116, such as the active sensor 120 and the controlsensor 122 in step 1F. The analyzer 118, the reader 114, or acombination thereof can monitor the electrical characteristic 800 of theactive sensor 120 upon introducing the sample effluent 134 containingthe anti-infective 104 to the active sensor 120. In addition, theanalyzer 118, the reader 114, or a combination thereof can also monitorthe electrical characteristic 800 of the control sensor 122 uponintroducing the sample effluent 134 without the anti-infective 104 tothe control sensor 122. The analyzer 118, the reader 114, or acombination thereof can compare the electrical characteristic 800 of theactive sensor 120 with the electrical characteristic 800 of the controlsensor 122 to assess the susceptibility of the infectious agent 102 tothe anti-infective 104.

The electrical characteristic 800 of the sensors 116 can differ when thesolution characteristic 136 of the sample effluents 134 differ as aresult of differences in the concentration or the amount of solutespresent in the sample effluents 134. For example, the electricalcharacteristic 800 of the active sensor 120 and the control sensor 122can differ when the solution characteristic 136 of the sample effluent134 introduced to the active sensor 120 differ from the solutioncharacteristic 136 of the sample effluent 134 introduced to the controlsensor 122. As a more specific example, the electrical characteristic800 of the active sensor 120 and the control sensor 122 can differ whenthe solution characteristic 136 of the sample effluents 134 differ intheir pH or differ in the concentration of another ion, an organicmolecule, or a combination thereof.

In another embodiment contemplated but not shown in FIG. 1, the analyzer118, the reader 114, or a combination thereof can monitor the electricalcharacteristic 800 of one sensor 116 upon introducing the sampleeffluent 134 without the anti-infective 104 to the sensor 116. In thisembodiment, additional nutrient solution 130 comprising theanti-infective 104 can be introduced or exposed to the filter surface126 comprising the infectious agent 102 and additional sample effluent134 resulting from this exposure step can be introduced to the sensor116. The analyzer 118, the reader 114, or a combination thereof candetect any changes in the electrical characteristic 800 of the sensor116 after introducing the additional sample effluent 134 to the sensor116. The analyzer 118, the reader 114, or a combination thereof can thenassess the susceptibility of the infectious agent 102 to theanti-infective 104 using any detected changes in the electricalcharacteristic 800 of the sensor 116.

In this embodiment, the change in the electrical characteristic 800 ofthe sensor 116 can indicate a change in the solution characteristic 136of the sample effluent 134 introduced to the sensor 116. For example,the change in the solution characteristic 136 of the sample effluent 134can indicate a change in the concentration of an ion, an organicmolecule, or a combination thereof in the sample effluent 134. As a morespecific example, the change in the solution characteristic 136 of thesample effluent 134 can be a change in the pH of the sample effluent134.

In these and other embodiments, the analyzer 118, the reader 114, or acombination thereof can assess the susceptibility of the infectiousagent 102 to the anti-infective 104 within a detection period 138. Inone embodiment, the detection period 138 can range from 60 minutes to240 minutes. In another embodiment, the detection period 138 can be lessthan 60 minutes. In yet another embodiment, the detection period 138 canbe greater than 240 minutes.

The reader 114 can produce an output signal 808 (see FIG. 8) assessingthe susceptibility of the infectious agent 102. In one embodiment, theoutput signal 808 can be an electrical signal. In this embodiment, theoutput signal 808 can be rendered as a graphic, such as a text string, anumber, a symbol, or a combination thereof on a display unit of thereader 114. In another embodiment, the output signal 808 can be an audiosignal.

The analyzer 118, the reader 114, or a combination thereof can assessthe susceptibility of the infectious agent 102 to the anti-infective 104as a binary assessment or a gradated or tiered assessment. In oneembodiment, the analyzer 118, the reader 114, or a combination thereofcan assess the susceptibility of the infectious agent 102 as eitherresistant or non-resistant to the anti-infective 104. In thisembodiment, the system 100 can introduce a set amount of theanti-infective 104 to the nutrient solution 130 and the reader 114 orthe analyzer 118 can assess the susceptibility of the infectious agent102 as either resistant or non-resistant based on any detected changesin the electrical characteristic 800 of one sensor 116 or any detecteddifferences in the electrical characteristic 800 of the active sensor120 and the control sensor 122.

For example, the reader 114, the analyzer 118, or a combination thereofcan assess the susceptibility of the infectious agent 102 as resistantto the anti-infective 104 when the analyzer 118 detects a change in theelectrical characteristic 800 of the one sensor 116 even afteranti-infective 104 is introduced to the filter surface 126 comprisingthe infectious agent 102. Also, for example, the reader 114, theanalyzer 118, or a combination thereof can assess the susceptibility ofthe infectious agent 102 as not resistant to the anti-infective 104 whenthe analyzer 118 fails to detect a change in the electricalcharacteristic 800 of the one sensor 116 when anti-infective 104 isintroduced to the filter surface 126 comprising the infectious agent102. Moreover, the reader 114, the analyzer 118, or a combinationthereof can assess the susceptibility of the infectious agent 102 as notresistant to the anti-infective 104 when the analyzer 118 fails todetect a statistically significant change or a change in the electricalcharacteristic 800 of the one sensor 116 exceeding a threshold value.

As another example, the reader 114, the analyzer 118, or a combinationthereof can assess the susceptibility of the infectious agent 102 asresistant to the anti-infective 104 when the analyzer 118 or the reader114 fails to detect a statistically significant difference between theelectrical characteristic 800 of the active sensor 120 and the controlsensor 122. More specifically, this statistically significant differencein the electrical characteristic 800 can be a difference exceeding athreshold value. In this example, the system 100 can introduce thesample effluent 134 from the nutrient solution 130 comprising theanti-infective 104 to the active sensor 120 and the sample effluent 134free from anti-infective 104 to the control sensor 122. In addition, thereader 114, the analyzer 118, or a combination thereof can assess thesusceptibility of the infectious agent 102 as not resistant to theanti-infective 104 when the reader 114 or the analyzer 118 detects astatistically significant difference between the electricalcharacteristic 800 of the active sensor 120 and the control sensor 122over time.

In other embodiments, the reader 114, the analyzer 118, or a combinationthereof can assess the level of susceptibility of the infectious agent102 on a gradated or tiered scale. For example, the reader 114 canassess the susceptibility of the infectious agent 102 as beingresistant, mildly susceptible, or susceptible to the anti-infective 104.In these embodiments, anti-infectives 104 of different concentrationscan be introduced to the filter surface 126 comprising the infectiousagent 102 to assess the level of susceptibility of the infectious agent102 to the anti-infective 104.

As a more specific example, when only one sensor 116 is used to assessthe level of susceptibility of the infectious agent 102, the system 100can introduce larger amounts of the anti-infective 104 to the filtersurface 126 over time and monitor the effects of the additionalanti-infective 104 on the electrical characteristic 800 of the sensor116 over such a time period. As another example, when multiple activesensors 120 are disposed on the substrate 112, the system 100 canintroduce differing amounts of the anti-infective 104 to differentactive sensors 120 simultaneously or over time and the reader 114, theanalyzer 118, or a combination thereof can compare the electricalcharacteristic 800 of the various active sensors 120 with the controlsensor 122 to assess the level of susceptibility of the infectious agent102 to the anti-infective 104.

While three categories of susceptibility are discussed in the sectionabove, it should be understood by one of ordinary skill in the art thatfour or greater categories of susceptibility can be used to assess thelevel of susceptibility of the infectious agent 102 to theanti-infective 104.

FIG. 2 illustrates another embodiment of the system 100 for detecting orassessing the susceptibility of an infectious agent 102 to ananti-infective 104. The system 100 can comprise the fluid deliverydevice 106, the substrate 112 comprising substrate wells 200, and thereader 114. The substrate 112 can have one or more sensors 116 disposedon a substrate surface 202. The system 100 can also include the analyzer118. In the embodiment shown in FIG. 2, the analyzer 118 can be disposedon the substrate surface 202. In other embodiments, the analyzer 118 canbe a standalone unit or device coupled to the substrate 112.

The sensors 116 can include one or more active sensors 120, one or morecontrol sensors 122, or a combination thereof disposed on the substratesurface 202. As illustrated in the embodiment shown in FIG. 2, theactive sensors 120 and control sensors 122 can be disposed on one sideof the substrate 112. In other embodiments not shown in FIG. 2, theactive sensors 120 and the control sensors 122 can be disposed ondifferent sides of the substrate 112 or on different substrates. Forexample, FIG. 2 shows the substrate 112 having three sensors 116;however, it is contemplated that the substrate 112 can comprise anynumber of sensors 116. In one embodiment, at least one of the sensors116 can be ISFET.

The substrate wells 200 can include a sample well 204, one or moreactive wells 206, one or more control wells 208, or a combinationthereof. The sample well 204, the one or more active wells 206, the oneor more control wells 208, or a combination thereof can be fluidlycoupled to or be in fluid communication with one another throughsubstrate channels 210. The substrate channels 210 can include tubes,capillaries, microfluidic channels, indentations, or holes disposed onor inside the substrate 112.

The substrate wells 200 including the sample well 204, the active well206, the control wells 208, or a combination thereof can be divots,indentations, or openings on the surface of the substrate 112. Inanother embodiment, the substrate wells 200 can be enclosed spaceswithin the substrate 112. In other embodiments, the substrate wells 200can be receptacles or cartridges coupled to the substrate 112. Thesubstrate wells 200 can also be fluidly coupled to or be in fluidcommunication with the sensors 116 through the substrate channels 210.

As illustrated in FIG. 2, the fluid delivery device 106 can deliver orinject the fluid sample 124 into the sample well 204 in step 2A. Thefluid sample 124 can comprise the infectious agent 102.

In another embodiment not shown in FIG. 2, a stimulus solution can beadded to the fluid sample 124 before introducing the fluid sample 124 tothe filter 110. The stimulus solution can be a nutrient or growthsolution. The stimulus solution can have a different composition thanthe nutrient solution 130. The stimulus solution can be a super nutrientsolution.

In an alternative embodiment not shown in FIG. 2, the fluid sample 124can be pre-filtered in a step before step 2A. This pre-filtering stepcan involve filtering the fluid sample 124 using the filter 110, amicrofluidic filter, or a combination thereof to filter out other largercellular components including blood cells or epithelial cells from thefluid sample 124.

The same fluid delivery device 106 or another fluid delivery device 106can also be used to deliver or inject the nutrient solution 130 to thesample well 204 in step 2B. The fluid delivery device 106 cancontinuously or periodically introduce or expose the substrate surface202 of the sample well 204 with the nutrient solution 130. In oneembodiment, the nutrient solution 130 can be composed of a buffercontaining bacto-tryptone, yeast extract, sodium chloride and anycombinations thereof. In another embodiment the nutrient solution caninclude a growth inducer. The growth inducer can be selected from thegroup consisting of a carbon-based inducer, a nitrogen-based inducer, amineral, a trace element, a biological growth factor, or any combinationthereof. For example, the growth inducer can include but is not limitedto glucose, ammonia, magnesium, or a combination thereof. For example,the nutrient solution 130 can be comprised of Tryptone, yeast extract,NaCl, and glucose.

The flow of the nutrient solution 130 can carry or deliver theinfectious agent 102 in the sample well 204 to the active well 206, thecontrol well 208, or a combination thereof. For example, the sample well204, the active well 206, the control well 208, or a combination thereofcan be shaped as a hemisphere having a rounded bottom, a cuboid having aflat or planar bottom, a cone, a frustoconical, a hyperboloid, or acombination thereof. The entire substrate 112 can be heated to atemperature between 30° C. to 40° C. when the infectious agent 102 is inthe active well 206, the control well 208, or a combination thereof andallowed to incubate for the incubation period 132. The substrate 112 canbe allowed to incubate in order to promote the proliferation,metabolism, or growth of the infectious agent 102 in the active wells206, the control wells 208, or a combination thereof.

The substrate wells 200, including the sample well 204, the active well206, the control well 208, or a combination thereof, can be covered by awell coating 212. The well coating 212 can cover or coat the bottom orsides of the wells. The well coating 212 can include an anti-buffercoating such as an acidic coating or a basic coating.

The well coating 212 can also be a trapping coating configured to trapthe infectious agent 102 in the active wells 206, the control wells 208,or a combination thereof. For example, the well coating 212 can be anextracellular matrix comprising proteins such as fibronectin, collagen,laminin, osteopontin, poly-D-lysine, or a combination thereof. The wellcoating 212 can also be a charged coating such as an amine surface, acarboxyl surface, a charged peptide surface, or a combination thereof.The well coating 212 can also be an oxygen or nitrogen containingsurface. The well coating 212 can also be a polyurethane surface.

The active wells 206, the control wells 208, or a combination thereofcan have a physical barrier 214. The physical barrier 214 can be aphysical feature or design of the well for trapping or isolating theinfectious agent 102 in the active well 206, the control well 208, or acombination thereof. For example, the physical barrier 214 can be anoverhang or lip protruding from a downstream section of the active well206, the control well 208, or a combination thereof. As another example,the physical barrier 214 can be a sloping surface of the active well206, the control well 208, or a combination thereof. In anotherembodiment contemplated but not shown in FIG. 2, the physical barrier214 can be the filter 110 disposed at an opening of the active well 206,the control well 208, or a combination thereof downstream from thesample well 204.

Although the example embodiment in FIG. 2 shows the physical barrier 214as a feature of the substrate wells 200, the physical barrier 214 canalso be a feature of the substrate channels 210. For example, thesubstrate channels 210 can be microfluidic channels, which narrow to awidth or diameter which prevent the infectious agent 102 from proceedingdown the substrate channels 210 toward the sensors 116. In this exampleembodiment, the substrate 112 can act as a microfluidic chip orlab-on-chip (LOC).

The well coating 212, the physical barrier 214, or a combination thereofcan be included as part of the system 100 to prevent or stop theinfectious agent 102 from contacting or reaching the sensors 116. Inanother embodiment contemplated but not shown in FIG. 2, an electricalor magnetic component can be used to trap or isolate the infectiousagent 102 in the active well 206, the control well 208, or a combinationthereof.

The nutrient solution 130 delivered in step 2B or additional nutrientsolution 130 can be continuously or periodically delivered or injectedinto the sample well 204, the active well 206, the control well 208, ora combination thereof until the infectious agent 102 is carried ordelivered into one or more active wells 206, control wells 208, or acombination thereof. The active wells 206, the control wells 208, or acombination thereof can comprise one or more openings, physicalfeatures, geometries, or device features which allow fluid orsupernatant in the active wells 206, the control wells 208, or acombination thereof to evacuate or exit the wells into one or moresubstrate channels 210. The fluid or supernatant sampled or separatedfrom the infectious agent 102 in the active wells 206, the control wells208, or a combination thereof can be referred to as the sample effluent134.

As illustrated in the example embodiment shown in FIG. 2, the sampleeffluent 134 can be introduced, carried, or delivered to one or more ofthe sensors 116 disposed on the substrate 112. The sample effluent 134can comprise a solution characteristic 136. The solution characteristic136 can include an amount or concentration of ions, organic moleculessuch as amino acids, minerals, or other inorganic compounds in thesample effluent 134.

The solution characteristic 136 can vary as a result of ions, organicmolecules, or minerals produced by or attributed to the infectious agent102 in the active wells 206, the control wells 208, or a combinationthereof. The solution characteristic 136 can be a direct or indirectbyproduct of a cellular activity undertaken by the infectious agent 102such as cell metabolism or cell growth. The sample effluent 134 cancomprise H⁺, ATP, CO₂, lactic acid, carbonic acid, NO₃ ⁻, or acombination thereof.

The substrate channels 120 can deliver or introduce sample effluent 134from one or more active wells 206 to one or more active sensors 120. Inaddition, separate substrate channels 120 can deliver or introducesample effluent 134 from one or more control wells 208 to one or morecontrol sensors 122.

After or prior to incubating the substrate 112, the same fluid deliverydevice 106 or another fluid delivery device 106 can be used to introducean anti-infective 104 to the active wells 206 in a step 2C. In theexample embodiment shown in FIG. 2, the anti-infective 104 can be mixedwith additional nutrient solution 130 and the active wells 206comprising the infectious agent 102 can be exposed to additionalnutrient solution 130 comprising the anti-infective 104. In otherembodiments, the anti-infective 104 can be introduced to the activewells 206 separate from the nutrient solution 130.

In the example embodiment shown in FIG. 2, nutrient solution 130containing the anti-infective 104 can be delivered or introduced to theactive well 206 comprising the infectious agent 102 while nutrientsolution 130 lacking the anti-infective 104 can be delivered orintroduced to the control well 208 also comprising the infectious agent102. In these embodiments, the sample effluent 134 flowing from theactive well 206 can be introduced to the active sensor 120 and thesample effluent 134 flowing from the control well 208 can be introducedto the control sensor 122.

In an alternative embodiment contemplated but not shown in FIG. 2, oneactive well 206 can initially be exposed to nutrient solution 130lacking in anti-infective 104 and the sample effluent 134 flowing fromthe active well 206 can be introduced to a sensor 116. In thisembodiment, the same active well 206 can be exposed at a later time withnutrient solution 130 comprising the anti-infective 104. By doing so,the sample effluent 134 from this second exposure step can be introducedto the same sensor 116 as the sensor 116 used to measure thenon-anti-infective sample effluent 134.

While FIG. 2 illustrates two of the three sensors 116 on the substrate112 being used to analyze sample effluent 134 from the fluid sample 124,it is contemplated that the substrate 112 can accommodate any number ofsensors 116 for receiving the sample effluent 134. For example, thesubstrate 112 can be a support or housing for a high throughput assayplate such as a 96-well plate, a 192-well plate, or a 384-well plate. Inthis example, each of the well plates can be in fluid communication withat least one sensor 116.

The reader 114, the analyzer 118, or a combination thereof can beconfigured to monitor the electrical characteristic 800 of the sensors116 upon introducing the sample effluent 134 to the sensors 116. Forexample, the reader 114 can monitor the electrical characteristic 800 ofthe sensors 116 by receiving one or more signals from the analyzer 118disposed on the substrate 112.

In the example embodiment shown in FIG. 2, the analyzer 118, the reader114, or a combination thereof can monitor the electrical characteristic800 of the sensors 116, such as the active sensor 120 and the controlsensor 122 in step 2D. The analyzer 118, the reader 114, or acombination thereof can monitor the electrical characteristic 800 of theactive sensor 120 upon introducing the sample effluent 134 from theactive well 206 to the active sensor 120. In addition, the analyzer 118,the reader 114, or a combination thereof can also monitor the electricalcharacteristic 800 of the control sensor 122 upon introducing the sampleeffluent 134 from the control well 208 to the control sensor 122. Theanalyzer 118, the reader 114, or a combination thereof can compare theelectrical characteristic 800 of the active sensor 120 with theelectrical characteristic 800 of the control sensor 122 to assess thesusceptibility of the infectious agent 102 to the anti-infective 104.

The electrical characteristic 800 of the sensors 116 can differ when thesolution characteristic 136 of the sample effluents 134 differ as aresult of differences in the concentration or the amount of solutespresent in the sample effluents 134. For example, the electricalcharacteristic 800 of the active sensor 120 and the control sensor 122can differ when the solution characteristic 136 of the sample effluent134 introduced to the active sensor 120 differ from the solutioncharacteristic 136 of the sample effluent 134 introduced to the controlsensor 122.

In another embodiment contemplated but not shown in FIG. 2, the analyzer118, the reader 114, or a combination thereof can monitor the electricalcharacteristic 800 of one sensor 116 upon introducing the sampleeffluent 134 without the anti-infective 104 to the sensor 116. In thisembodiment, additional nutrient solution 130 comprising theanti-infective 104 can be delivered or exposed to the same sensor 116and additional sample effluent 134 resulting from this exposure step canbe introduced to the sensor 116. The analyzer 118, the reader 114, or acombination thereof can detect any changes in the electricalcharacteristic 800 of the sensor 116 after introducing the additionalsample effluent 134 to the sensor 116. The analyzer 118, the reader 114,or a combination thereof can then assess the susceptibility of theinfectious agent 102 to the anti-infective 104 using any detectedchanges in the electrical characteristic 800 of the sensor 116.

In this embodiment, the change in the electrical characteristic 800 ofthe sensor 116 can indicate a change in the solution characteristic 136of the sample effluent 134 introduced to the sensor 116. For example,the change in the solution characteristic 136 of the sample effluent 134can indicate a change in the concentration of an ion, an organicmolecule, or a combination thereof in the sample effluent 134. As a morespecific example, the change in the solution characteristic 136 of thesample effluent 134 can be a change in the pH of the sample effluent134.

In these and other embodiments, the analyzer 118, the reader 114, or acombination thereof can assess the susceptibility of the infectiousagent 102 to the anti-infective 104 within the detection period 138.

The reader 114 can also produce the output signal 808 assessing thesusceptibility of the infectious agent 102. The analyzer 118, the reader114, or a combination thereof can assess the susceptibility of theinfectious agent 102 to the anti-infective 104 as the binary assessmentor the gradated or tiered assessment.

For example, the reader 114, the analyzer 118, or a combination thereofcan assess the susceptibility of the infectious agent 102 as resistantto the anti-infective 104 when the analyzer 118 detects a change in theelectrical characteristic 800 of the active sensor 120 even afteranti-infective 104 is introduced to the active well 206 fluidly coupledto the active sensor 120. Also, for example, the reader 114, theanalyzer 118, or a combination thereof can assess the susceptibility ofthe infectious agent 102 as not resistant to the anti-infective 104 whenthe analyzer 118 fails to detect a change in the electricalcharacteristic 800 of the active sensor 120 when anti-infective 104 isintroduced to the active well 206 fluidly coupled to the active sensor120. Moreover, the reader 114, the analyzer 118, or a combinationthereof can assess the susceptibility of the infectious agent 102 as notresistant to the anti-infective 104 when the analyzer 118 fails todetect a statistically significant change or a change in the electricalcharacteristic 800 of the active sensor 120 exceeding a threshold value.

As another example, the reader 114, the analyzer 118, or a combinationthereof can assess the susceptibility of the infectious agent 102 asresistant to the anti-infective 104 when the analyzer 118 or the reader114 fails to detect a statistically significant difference between theelectrical characteristic 800 of the active sensor 120 and the controlsensor 122. More specifically, a statistically significant difference inthe electrical characteristic 800 can be a difference exceeding athreshold value. In this example, the system 100 can introduce thesample effluent 134 from the active well 206 to the active sensor 120and the sample effluent 134 from the control well 208 to the controlsensor 122. In addition, the reader 114, the analyzer 118, or acombination thereof can assess the susceptibility of the infectiousagent 102 as not resistant to the anti-infective 104 when the reader 114or the analyzer 118 detects a statistically significant differencebetween the electrical characteristic 800 of the active sensor 120 andthe control sensor 122.

In other embodiments, the reader 114, the analyzer 118, or a combinationthereof can assess the level of susceptibility of the infectious agent102 on a gradated or tiered scale. For example, the reader 114, theanalyzer 118, or a combination thereof can assess the susceptibility ofthe infectious agent 102 as being resistant, mildly susceptible, orsusceptible to the anti-infective 104. In these embodiments,anti-infectives 104 of different concentrations can be introduced todifferent active wells 206 comprising the infectious agent 102 to assessthe level of susceptibility of the infectious agent 102 to theanti-infective 104.

As an example, when only one sensor 116 is used to assess the level ofsusceptibility of the infectious agent 102, the system 100 can introducelarger amounts of the anti-infective 104 to the active well 206 overtime and monitor the effects of the additional anti-infective 104 on theelectrical characteristic 800 of the active sensor 120 fluidly coupledto the active well 206 over such a time period. As another example, whenmultiple active sensors 120 are disposed on the substrate 112, thesystem 100 can introduce differing amounts of the anti-infective 104 todifferent active wells 206 and the reader 114, the analyzer 118, or acombination thereof can compare the electrical characteristic 800 of thevarious active sensors 120 with one or more control sensors 122 toassess the level of susceptibility of the infectious agent 102 to theanti-infective 104.

FIG. 3 illustrates another embodiment of the system 100 for detecting orassessing the susceptibility of an infectious agent 102 to ananti-infective 104. The system 100 can comprise the fluid deliverydevice 106, the filter housing 108 containing the filter 110, and asensor device 300. In one embodiment, the sensor device 300 can be ahandheld ISFET meter or probe.

As illustrated in FIG. 3, the fluid delivery device 106 can deliver orinject the fluid sample 124 into the filter housing 108 in step 3A. Thefluid sample 124 can comprise the infectious agent 102. In the exampleembodiment shown in FIG. 3, the fluid delivery device can be a pump. Forexample, the fluid delivery device 106 can be a syringe pump, apneumatic pump, or a hydraulic pump.

In other embodiments not shown in FIG. 3, the fluid delivery device 106can be an injection cartridge, a microfluidic device, a pipette, areaction tube, a capillary, a test tube, a combination thereof, or aportion therein.

The filter housing 108 can be a container or vessel configured to secureor enclose the filter 110. The filter housing 108 can be a container orvessel configured to secure or enclose the filter 110. The filterhousing 108 can also be a protective chamber. The protective chamber canbe an electrically isolated environment, a temperature controlledchamber, and/or a light controlled chamber. For example, the filterhousing 108 can be a housing of a syringe filter.

The filter 110 can be a non-clogging filter. The filter 110 can have anon-clogging filter surface. The filter 110 can also have filter poresof sequentially smaller pore size. For example, the filter 110 can havelarger filter pores at the top of the filter and progressively smallerfilters pores toward the bottom of the filter 110. Although not shown inFIG. 3, it is contemplated by this disclosure that the filter 110 canrefer to a plurality of filters in a stacked arrangement.

The filter 110 can be a mesh or matrix for isolating or separating theinfectious agent 102 or other molecules or cells from the supernatant ofthe fluid sample 124.

The filter 110 can comprise a filter surface 126. The filter surface 126can be the portion of the filter 110 used to isolate or trap theinfectious agent 102. The filter surface 126 can include an externalsurface, an internal surface extending into the filter 110, or acombination thereof. Although not shown in FIG. 3, the filter housing108 can have at least one opening 128 to allow fluid or supernatant fromthe fluid sample 124 to evacuate the filter housing 108. For example,step 3A can include the additional step of discarding the fluid orsupernatant from the fluid sample 124 through the opening 128 afterisolating the infectious agent 102 on the filter surface 126.

In another embodiment not shown in FIG. 3, a stimulus solution can beadded to the fluid sample 124 before introducing the fluid sample 124 tothe filter 110. The stimulus solution can be a nutrient or growthsolution. The stimulus solution can have a different composition thanthe nutrient solution 130. The stimulus solution can be a super nutrientsolution.

In an alternative embodiment not shown in FIG. 3, the fluid sample 124can be pre-filtered in a step before step 3A. This pre-filtering stepcan involve filtering the fluid sample 124 using another instance of thefilter 110, a microfluidic filter, or a combination thereof to filterout other larger cellular components including blood cells or epithelialcells from the fluid sample 124 when the fluid sample 124 is composed ofa bodily fluid or sample.

The same fluid delivery device 106 or another fluid delivery device 106can also be used to deliver or inject a nutrient solution 130 to thefilter housing 108 in step 3B. The fluid delivery device 106 cancontinuously or periodically introduce or expose the nutrient solution130 to the filter surface 126 containing the infectious agent 102. Inone embodiment, the nutrient solution 130 can be composed of Tryptone,yeast extract, NaCl, glucose, and the buffer.

The filter housing 108 comprising the nutrient solution 130, the filter,and the infectious agent 102 can be heated to a temperature of around37° C. and allowed to incubate for an incubation period 132 in a step3C. The incubation period 132 can range from 15 minutes to one hour. Inother embodiments, the incubation period 132 can be less than 15minutes. The incubation period 132 can be adjusted based on the type ofinfectious agent 102.

The incubation period 132 can also be adjusted based on the amount ofthe infectious agent 102 present in the fluid sample 124. For example,the incubation period 132 can be increased when the amount of theinfectious agent 102 is below a threshold amount. The filter 110 can beallowed to incubate with the nutrient solution 130 in order to promotethe metabolism of the infectious agent 102 on the filter 110.Furthermore, by monitoring the rate at which metabolites are producedusing the sensor herein described, it is possible to identify theinfectious agent, as different infectious agents have characteristicrates of multiplication and metabolism. There is an additional featurehereby disclosed, namely providing different nutrients to the infectiousagents over time while monitoring the rate of production of variousmetabolites using the sensor herein described in order to furtheridentify the infectious agent.

After incubating the filter housing 108, the filter 110 comprising theinfectious agent 102 can be separated from a solution representing theleftover nutrient solution 130 in the filter housing 108. This solutioncan be referred to as the sample effluent 134. The sensor device 300 canthen be introduced or inserted in to the sample effluent 134 in step 3Dto determine the solution characteristic 136 of the sample effluent 134.In another embodiment contemplated but not shown in FIG. 3, the sampleeffluent 134 can be evacuated or removed from the filter housing 108through an opening in the filter housing 108 into another container orvessel. The sensor device 300 can then be used to determine the solutioncharacteristic 136 of the sample effluent 134 in this other container orvessel.

The solution characteristic 136 can refer to one or more attributes ofthe solution making up the sample effluent 134. For example, thesolution characteristic 136 can include a concentration of a solute oran absolute number of solute molecules in solution. The solutioncharacteristic 136 can include an amount or concentration of ions,organic molecules such as amino acids, minerals, or other inorganiccompounds in the sample effluent 134.

The solution characteristic 136 can vary as a result of ions, organicmolecules, or minerals produced by or attributed to the infectious agent102 on the filter 110. The solution characteristic 136 can be a director indirect byproduct of a cellular activity undertaken by theinfectious agent 102 such as cell metabolism or cell growth. In oneembodiment, the sample effluent 134 can comprise hydrogen ions (H⁺) as abyproduct of bacterial cell metabolism or growth. In other embodiments,the sample effluent 134 can comprise adenosine triphosphate (ATP),carbon dioxide (CO₂), lactic acid, carbonic acid, nitrates (NO₃ ⁻), acombination thereof, or any other metabolic byproduct produced by orattributed to the infectious agent 102.

After step 3C, the filter 110 comprising the infectious agent 102 can beremoved from the filter housing 108 containing the sample effluent 134and placed into a new filter housing 108. The same fluid delivery device106 or another fluid delivery device 106 can then be used to introducean anti-infective 104 to the new filter housing 108 containing thefilter 110 in a step 3E. In an alternative embodiment, step 3E caninvolve using the same fluid delivery device 106 or another fluiddelivery device 106 to introduce an anti-infective 104 to the filterhousing 108 from step 3C after the sample effluent 134 has beenevacuated or removed from the opening of the filter housing 108.

In the example embodiment shown in FIG. 3, the anti-infective 104 can bemixed with additional nutrient solution 130 and the filter 110comprising the infectious agent 102 can be exposed to additionalnutrient solution 130. In other embodiments, the anti-infective 104 canbe introduced to the filter 110 separate from the nutrient solution 130.

After introducing the anti-infective 104 to the filter housing 108, thefilter housing 108 comprising the nutrient solution 130, the filter 110,the anti-infective 104, and the infectious agent 102 can be heated to atemperature of around 37° C. and allowed to incubate for an incubationperiod 132 in a step 3F.

After incubating the filter housing 108, the filter 110 comprising theinfectious agent 102 can be separated from the sample effluent 134. Asensor device 300 can then be introduced or inserted into the sampleeffluent 134 in step 3G to determine the solution characteristic 136 ofthe sample effluent 134. In another embodiment contemplated but notshown in FIG. 3, the sample effluent 134 can be evacuated or removedfrom the filter housing 108 through an opening in the filter housing 108into another container or vessel. The sensor device 300 can then be usedto determine the solution characteristic 136 of the sample effluent 134in this other container or vessel.

The reader 114 can then be used to compare the solution characteristic136 of the sample effluent 134 from step 3G with the solutioncharacteristic 136 of the sample effluent 134 from step 3D to assess thesusceptibility of the infectious agent 102 to the anti-infective 104 instep 3H. For example, the reader 114 can be used to compare the twosolution characteristics 136 over time. The solution characteristic 136from step 3D and step 3G can differ as a result of differences in theconcentration or the amount of solutes present in the sample effluents134. For example, the solution characteristic 136 can differ in their pHor differ in the concentration of another ion, an organic molecule, or acombination thereof.

The reader 114 can assess the susceptibility of the infectious agent 102to the anti-infective 104 within a detection period 138. In oneembodiment, the detection period 138 can range from 60 minutes to 240minutes. In another embodiment, the detection period 138 can be lessthan 60 minutes. In yet another embodiment, the detection period 138 canbe greater than 240 minutes.

The reader 114 can produce an output signal 808 assessing thesusceptibility of the infectious agent 102. In one embodiment, theoutput signal 808 can be an electrical signal. In this embodiment, theoutput signal 808 can be rendered as a graphic, such as a text string, anumber, a symbol, or a combination thereof on a display unit of thereader 114. In another embodiment, the output signal 808 can be an audiosignal.

The reader 114 can assess the susceptibility of the infectious agent 102to the anti-infective 104 as a binary assessment or a gradated or tieredassessment. In one embodiment, the reader 114 can assess thesusceptibility of the infectious agent 102 as either resistant ornon-resistant to the anti-infective 104. In this embodiment, the reader114 can assess the susceptibility of the infectious agent 102 as eitherresistant or non-resistant based on any detected differences in thesolution characteristic 136.

For example, the reader 114 can assess the susceptibility of theinfectious agent 102 as resistant to the anti-infective 104 when thereader 114 fails to detect a statistically significant differencebetween the solution characteristic 136 from step 3D and the solutioncharacteristic 136 from step 3G over time. A statistically significantdifference can refer to a difference exceeding a threshold value. Also,the reader 114 can assess the susceptibility of the infectious agent 102as sensitive to the anti-infective 104 when the reader 114 detects astatistically significant difference between the solution characteristic136 from step 3D and the solution characteristic 136 from step 3G overtime.

In other embodiments, the reader 114 can assess the level ofsusceptibility of the infectious agent 102 on a gradated or tieredscale. For example, the reader 114 can assess the susceptibility of theinfectious agent 102 as being resistant, mildly susceptible, orsusceptible to the anti-infective 104. In these embodiments,anti-infectives 104 of different concentrations can be introduced to thefilter housing 108 in step 3E to assess the level of susceptibility ofthe infectious agent 102 to the anti-infective 104. The reader 114 cancompare the solution characteristic 136 of the various sample effluents134 over time to assess the level of susceptibility of the infectiousagent 102 to the anti-infective 104.

FIG. 4A illustrates a side view of an active sensor 120. The activesensor 120 can be disposed on a semiconductor layer 404. The activesensor 120 can have an external reference electrode 400 extending into ameasured liquid 402 in contact with the active sensor 120. As depictedin FIG. 4A, the active sensor 120 can comprise a semiconductor layer 404and a base dielectric layer 406. The active sensor 120 can comprise apolymer layer, a metal layer, a metalloid layer, a ceramic layer, anorganic semiconductor, a carbon nanotube layer, a graphene layer, anorganic conductor such as those derived from polyacetylene, polyaniline,Quinacridone, Poly(3,4-ethylenedioxythiophene) or PEDOT, PEDOT:polystyrene sulfonate (PSS), or a combination thereof. The semiconductorlayer 404 can be composed of silicon or an oxide of silicon which allowsa voltage to be applied through the semiconductor layer 404 to thesensor channel 410.

The base dielectric layer 406 can be coupled or can be disposed on thesemiconductor layer 404 to electrically insulate or isolate each of thesensors 116 from one another. In one embodiment, the base dielectriclayer 406 can be composed of an oxide material. In other embodiments,the base dielectric layer 406 can be composed of any other materialcapable of providing insulation.

In one or more embodiments, the sensors 116 of the system 100, includingthe active sensor 120, the control sensor 122, or a combination thereofcan be fabricated using a complementary metal oxide semiconductor (CMOS)process or a similar process. For example, the active sensor 120, thecontrol sensor 122, or a combination thereof can be integrated CMOSISFET sensors fabricated from p-type and n-type metal oxidesemiconductor field-effect transistors (MOSFETs). In another embodiment,the sensors 116 can be organic field-effect transistors (OFETs).

As depicted in FIG. 4A, the active sensor 120 can comprise sensorcontacts 408, a sensor channel 410 in between the sensor contacts 408, agate dielectric layer 412 coupled to or on top of the sensor channel410, and an encapsulating layer 414 partially covering the gatedielectric layer 412 of the active sensor 120. The sensor contacts 408can include a source contact and a drain contact. For example, thesensor contacts 408 can be composed of highly doped p-type material. Thesensor channel 410 can act as a bridge between the two sensor contacts408 and can be composed of any electrically conductive material orcoating that allows for electrical communication between the sensorcontacts 408.

The gate dielectric layer 412 can be coupled to or disposed on top ofthe sensor channel 410. In certain embodiments, the gate dielectriclayer 412 can be a high-k dielectric layer or a material layer having ahigh dielectric constant (k). For example, the gate dielectric layer 412can comprise aluminum oxide, hafnium oxide, titanium oxide, zirconiumoxide, yttrium oxide, tantalum oxide, hafnium silicate, zirconiumsilicate, silicon nitride, aluminum nitride, hafnium nitride, zirconiumnitride, or a combination thereof. As a more specific example, the gatedielectric layer 412 can comprise aluminum dioxide, hafnium dioxide,zirconium dioxide, or a combination thereof. In other embodiments, thegate dielectric layer 412 can comprise a silicon dioxide layer.

Any of the sensors 116, including the active sensor 120 and the controlsensor 122, can be partially covered by the encapsulating layer 414. Theencapsulating layer 414 can be composed of any inert or non-conductivematerial for protecting the sensor 116 from being exposed to solutes orcontaminants in the measured liquid 402 that would damage or degrade thesensor 116.

As depicted in FIG. 4A, the system 100 can also comprise an externalreference electrode 400 in liquid communication with the measured liquid402 and the sensor 116 itself. The measured liquid 402 can refer to anyof the sample effluent 134, the nutrient solution 130, the fluid sample124, a portion therein, or a combination thereof. The fluid sample 124can be introduced to the sensors 116 from the filter housing 108, thesubstrate wells 200, or any other fluid delivery device 106. The fluidsample 124 can cover the active sensor 120, the control sensor 122, or acombination thereof when introduced to the sensors 116. In otherembodiments, the fluid sample 124 can partially cover or be in liquidcommunication with the active sensor 120, the control sensor 122, or acombination thereof when introduced to the sensors 116.

The external reference electrode 400 can apply a potential, such as aliquid gate potential, to the measured liquid 402. In one embodiment,the external reference electrode 400 can be a standalone probe orelectrode. In other embodiments, the external reference electrode 400can be coupled to the reader 114, the analyzer 118, or a combinationthereof. The external reference electrode 400 can have a stable andwell-known internal voltage and can act as a differential noise filterfor removing electrical noise from measurements taken by the sensors116.

In one embodiment, the external reference electrode 400 can be asilver/silver chloride (Ag/AgCl) electrode. In other embodiments, theexternal reference electrode 400 can be a saturated calomel referenceelectrode (SCE) or a copper-copper (II) sulfate electrode (CSE).

The system 100 can use the external reference electrode 400 to determineor record a relative change in the electrical characteristic 800 of theactive sensor 120 rather than having to ascertain an absolute change.The system 100 can also use the external reference electrode 400 todetermine or record a relative difference between the electricalcharacteristic 800 of the active sensor 120 and the control sensor 122.

A back-gate voltage Vbg can be applied via the silicon substrate. Theelectrical characterization of ISFETs can be performed by applying asource-drain voltage Vsd to measure the source-drain current Isd. Inanother embodiment, a liquid gate voltage can be applied to a solutionvia a reference electrode. The electrical characterization of ISFETs canbe performed applying a source-drain voltage Vsd to measure thesource-drain current Isd. In another embodiment, a dual-gate approachcan be used by applying gate voltages simultaneously to the back gateand to the liquid gate. This allows an operator to tune the device todifferent working positions, optimizing the sensitivity. The back-gatevoltage Vbg is applied to the Si substrate, while the liquid gatevoltage VIg is applied via a reference electrode. At the same time, theliquid potential VIg can be measured by the reference electrode. Whenthe ion concentration in the solution is changing, the ISFET respondswith a change in the electrical characteristic. For example, in case ofproton (H+) changes, the protons interact with the oxide surface of theISFET. This is the expected dependence for an oxide surface exposinghydroxl (—OH) groups to the liquid. The change in surface charge densitycaused by a pH change is described by the site-binding model, whichtakes into account that —OH groups can be protonated or deprotonated.This model predicts an approximate linear relation between the surfacecharge density and the proton concentration. Since the surface chargeacts as an additional gate, the ISFET is responding to the additionalgate effect.

FIG. 4B illustrates a side view of another embodiment of thesemiconductor layer 404 having the active sensor 120 and an on-chipreference electrode 416 disposed on it. The on-chip reference electrode416 can serve the same purpose as the external reference electrode 400except fabricated as a chip or sensor on the semiconductor layer 404 orthe insulator layer 406. The on-chip reference electrode 416 can belocated adjacent to or near the active sensor 120. The on-chip referenceelectrode 416 can be on the base dielectric layer 406. The on-chipreference electrode 416 can also be partially covered by theencapsulating layer 414. The on-chip reference electrode 416 can apply aliquid gate voltage (V_(LG)) to the measured liquid 402.

The on-chip reference electrode 416, the external reference electrode400, or a combination thereof can be comprised of a metal, asemiconductor material, or a combination thereof. In one embodiment, thecontrol sensor 122 can act as the on-chip reference electrode 416. Themetal of the on-chip reference electrode 416 can be covered by an oxidelayer, a silane layer, or a combination thereof. Since metals or othermaterials used to fabricate such reference electrodes can often have aninhibitory or harmful effect on the infectious agents 102 underinvestigation, one advantage of the methods and systems 100 disclosedherein is the separation of the infectious agent 102 from the componentsof the system 100 in physical or fluid contact with these referenceelectrodes.

For example, the external reference electrode 400 can be an Ag/AgClreference electrode. In this example, silver ions or a silver surfacemaking up the external reference electrode 400 can act as ananti-infective agent when placed into contact with certain types ofbacteria, fungus, virus, or prion. By separating the sample effluent 134from the bacteria, fungus, virus, or prion representing the infectiousagent 102, the system 100 can prevent false positive or false negativeresults stemming from the antibacterial effects of the referenceelectrode on the infectious agent 102 under investigation. For example,the filter 110 or the substrate wells 200 can trap or isolate theinfectious agent 102 but permit the nutrient solution 130 or the sampleeffluent 134 to reach the sensors 116 and the reference electrode.

The on-chip reference electrode 416 can be a transistor with verysimilar electrical properties as compared to the sensor 116 but with apassivated surface, the so-called reference FET (RFET). The RFET can bean ISFET with a pH-passivating membrane, ion-blocking layers ofphotoresist material, or other polymers. The on-chip reference electrode416 can comprise one or more pH-insensitive layers covering an ISFET.Such pH-insensitive layers can include silanes, self-assembled monolayers (SAMs), buffered hydrogels, PVC, parylene, polyACE, or any otherchemically inert material. Also a metal, such as Ag or Pt, can be usedas a quasi-reference electrode evaporated on the substrate carrier. Inanother embodiment, the on-chip reference electrode 416 can be a metalcombined with a metal salt such as an Ag/AgCl reference electrode.

FIG. 5A illustrates a side view of yet another embodiment of thesemiconductor layer 404 having the active sensor 120 and a controlsensor 122 disposed on it and the external reference electrode 400extending into the measured liquid 402 in contact with the active sensor120 and the control sensor 122. Similar to the active sensor 120, thecontrol sensor 122 can comprise a pair of sensor contacts 408, a sensorchannel 410 in between the sensor contacts 408, a gate dielectric layer412 coupled to or on top of the sensor channel 410, and an encapsulatinglayer 414 partially covering the gate dielectric layer 412 of thecontrol sensor 122.

The sensor contacts 408 can include a source contact and a draincontact. The sensor channel 410 can act as a bridge between the twosensor contacts 408 and can be composed of any electrically conductivematerial or coating that allows for electrical communication between thesensor contacts 408.

The gate dielectric layer 412 can be coupled to or disposed on top ofthe sensor channel 410. In certain embodiments, the gate dielectriclayer 412 can be a high-k dielectric layer or a material layer having ahigh dielectric constant. For example, the gate dielectric layer 412 ofthe control sensor 122 can comprise aluminum oxide, hafnium oxide,titanium oxide, zirconium oxide, yttrium oxide, tantalum oxide, hafniumsilicate, zirconium silicate, or a combination thereof. As a morespecific example, the gate dielectric layer 412 can comprise aluminumdioxide, hafnium dioxide, zirconium dioxide, or a combination thereof.In other embodiments, the gate dielectric layer 412 can comprise asilicon dioxide layer.

The encapsulating layer 414 can be composed of any inert ornon-conductive material for protecting the control sensor 122 from beingexposed to solutes or contaminants in the measured liquid 402 that woulddamage or degrade the control sensor 122.

In the example embodiment shown in FIG. 5A, the control sensor 122 cancomprise a passivation layer 500 coupled to or disposed on the gatedielectric layer 412. The passivation layer 500 can be composed of apolymer layer, a metallic layer, a self-assembled monolayer (SAM), or acombination thereof. The passivation layer 500 can be used to preventbinding of ions or molecules to the surface of the control sensor 122.In other embodiments, the control sensor 122 can be without thepassivation layer 500 and the makeup of the control sensor 122 can beidentical to the active sensor 120. For example, the passivation layer500 can be a pH-passivating membrane, an ion-blocking layer, aphotoresist material, or any other polymer. In addition, the passivationlayer 500 can be a pH-insensitive layer covering an ISFET. Example ofpH-insensitive layers include silanes, SAMs, buffered hydrogels, PVC,parylene, polyACE, or a combination thereof.

FIG. 5B illustrates a side view of another embodiment of thesemiconductor layer 404 having the active sensor 120, the control sensor122, and the on-chip reference electrode 416 disposed on it. As shown inFIG. 5B, the on-chip reference electrode 416 can be disposed or locatedin between the active sensor 120 and the control sensor 122.

FIG. 6A illustrates a side view of an embodiment of the active sensor120 and the control sensor 122 each having an extended gate 600. Theextended gate 600 can be an extension of the gate dielectric layer 412.

FIG. 6B illustrates a side view of another embodiment of the activesensor 120 and the control sensor 122 each having the extended gate 600and an on-chip reference electrode 416 adjacent to the active sensor120. As shown in FIGS. 6A and 6B, only the extended gate is exposed tothe liquid. The extended gate can interact with particles in thesolution. The extended gate can reduce the amount of material needed tomake the active sensor 120.

FIG. 7 illustrates an embodiment of the system 100 fabricated ordesigned as a disposable strip 700. The disposable strip 700 cancomprise a number of active sensors 120 and control sensors 122 and ananalyzer 118 disposed on a strip substrate. In one embodiment, sampleeffluent 134 resulting from step 1E or step 1D depicted in FIG. 1 can beintroduced to one end of the disposable strip 700 and the other end ofthe disposable strip 700 can be electrically coupled to or fed into thereader 114. In another embodiment, a fluid sample 124 can be introducedto one end of the disposable strip 700 as shown in step 2A of FIG. 2 andsample effluent 134 can flow to the active sensors 120, the controlsensors 122, or a combination thereof on the disposable strip 700.Although not shown in FIG. 7, substrate wells 200 such as the activewells 206 and the control wells 208 of FIG. 2 can be disposed on thestrip substrate upstream from the sensors 116. The reader 114 can thenassess the susceptibility of an infectious agent 102 in the fluid sample124 to the anti-infective 104 introduced to or coated on the disposablestrip 700.

FIG. 8 illustrates one embodiment of the analyzer 118 and the reader 114processing signals outputted by the active sensor 120 and the controlsensor 122. The analyzer 118, the reader 114, or a combination thereofcan monitor the electrical characteristic 800 of the sensors 116including the active sensor 120, the control sensor 122, or acombination thereof.

The active sensor 120 can produce an active signal 802. The activesignal 802 can be indicative of a change in the electricalcharacteristic 800 of the active sensor 120. For example, the activesignal 802 can be indicative of a change in the current, the voltage,the threshold voltage, the capacitance, or the resistance of the activesensor 120. The active sensor 120 can exhibit a change in its electricalcharacteristic 800 due to a change in the solution characteristic 136 ofa measured liquid 402 contacting or introduced to the active sensor 120.For example, the active sensor 120 can exhibit a change in itselectrical characteristic 800 due to a change in the solutioncharacteristic 136 of the sample effluent 134 introduced to the activesensor 120. As a more specific example, the change in the solutioncharacteristic 136 can be change in the concentration of an ion or achange in the pH of the measured liquid 402 contacting or introduced tothe active sensor 120.

The control sensor 122 can produce a control signal 804. The controlsignal can be indicative of a change in the electrical characteristic800 of the control sensor 122. The control signal can be analyzedrelative to the reference electrode. For example, the control signal 804can be indicative of the change in the current, the voltage, thethreshold voltage, the capacitance, or the resistance of the controlsensor 122. Similar to the active sensor 120, the control sensor 122 canexhibit a change in its electrical characteristic 800 due to a change inthe solution characteristic 136 of a measured liquid 402 contacting orintroduced to the control sensor 122.

The analyzer 118 can receive as inputs the active signal 802 from theactive sensor 120 and the control signal 804 from the control sensor122. The analyzer 118 can produce a differential signal 806. In oneembodiment, the differential signal 806 can be a difference between theactive signal 802 and the control signal 804. The differential signal806 or ΔS can also be indicative of a change in the electricalcharacteristic 800 of the active sensor 120 or the control sensor 122 ora difference between the electrical characteristic 800 of the activesensor 120 and the control sensor 122. The reader 114 and the analyzer118 can also provide a feedback loop to control the active sensor 120.

The analyzer 118 can also convert the active signal 802 and the controlsignal 804 from analog to digital. The differential signal 806 can betransmitted to the reader 114, and used to assess the susceptibility ofthe infectious agent 102 in the fluid sample 124 to one or moreanti-infectives 104. The reader 114 can also provide an output signal808 assessing the susceptibility of the infectious agent 102 to one ormore anti-infectives 104. In one embodiment, the reader 114 can providean output signal 808 indicating whether the infectious agent 102 isresistant or sensitive to an anti-infective 104. In another embodiment,the reader 114 can provide an output signal 808 indicating a level ofsusceptibility of the infectious agent 102 to one or moreanti-infectives 104 such as susceptible, mildly susceptible, orresistant.

FIG. 9 illustrates experimental results of experiments conducted usingthe methods and system 100 described herein. The graphs in FIG. 9 show achange in the solution characteristic 136 of the measured liquid 402,such as the sample effluent 134, monitored by the system 100 at aspecific point in time. In this case, the graphs in FIG. 9 show thechange in the solution characteristic 136 of the measured liquid 402sixty (60) minutes after an anti-infective 104 is introduced to thefilter 110, the substrate wells 200, or a combination thereof comprisingthe infectious agent 102.

As shown in FIG. 9, the change in the solution characteristic 136 can bea change in the pH of the measured liquid 402. For example, the graphsin FIG. 9 show the effects of various anti-infectives 104 on E. coli. Inthis example, E. coli can be one of the infectious agents 102 present inthe fluid sample 124 applied or introduced to the system 100. As shownin FIG. 9, a change in the solution characteristic 136, such as a changein the pH, can indicate resistance of the infectious agent 102 to theanti-infective 104 while a lack of a change or an insignificant changein the solution characteristic 136 can indicate a susceptibility of theinfectious agent 102 to the anti-infective 104. An insignificant changein the solution characteristic 136 can be a change below a statisticallysignificant percentage or threshold value.

For example, one of the graphs shows the effects of the anti-infective104 nitrofurantoin on the pH of the measured liquid 402, such as thesample effluent 134, 60 minutes after E. coli from the fluid sample 124is exposed to nitrofurantoin of various concentrations. As can be seenin the graph, the E. coli in the fluid sample 124 can be resistant toapproximately 1 μg/ml of nitrofurantoin but can be susceptible whenexposed to approximately 10 μg/ml of nitrofurantoin.

Also, for example, another one of the graphs shows the effects of theanti-infective ciprofloxacin on the pH of the measured liquid 402, suchas the sample effluent 134, 60 minutes after E. coli from the fluidsample 124 is exposed to ciprofloxacin of various concentrations. Thisgraph shows that the systems, devices, and methods disclosed herein canbe used to determine the minimal inhibitory concentration (MIC) of ananti-infective on an infectious agent. As can be seen in the graph, theE. coli in the fluid sample 124 can be resistant to approximately 0.1μg/ml of ciprofloxacin but can be susceptible when exposed toapproximately 1 μg/ml of nitrofurantoin. In this case, 1 μg/ml can bethe MIC of nitrofurantoin on the E. coli isolated from the fluid sample124.

FIG. 10 illustrates additional experimental results of experimentsconducted using the methods and system 100 described herein. The graphsin FIG. 10 show the effects of various anti-infectives 104 on thebacteria Staphylococcus saprophyticus. In these examples, Staphylococcussaprophyticus can be one of the infectious agents 102 present in thefluid sample 124 applied or introduced to the system 100.

For example, one of the graphs shows the effects of the anti-infective104 ampicillin on the pH of the measured liquid 402, such as the sampleeffluent 134, 60 minutes after Staphylococcus saprophyticus from thefluid sample 124 is exposed to ampicillin of various concentrations. Ascan be seen in the graph, the Staphylococcus saprophyticus in the fluidsample 124 can be resistant to ampicillin when up to 50 μg/ml ofampicillin is introduced to filters or wells comprising the infectiousagent 102.

Also, for example, another one of the graphs shows the effects of theanti-infective nitrofurantoin on the pH of the measured liquid 402, suchas the sample effluent 134, 60 minutes after Staphylococcussaprophyticus from the fluid sample 124 is exposed to nitrofurantoin ofvarious concentrations. As can be seen in the graph, the Staphylococcussaprophyticus in the fluid sample 124 can be resistant to approximately1 μg/ml of nitrofurantoin but can be susceptible when exposed toconcentrations higher than 1 μg/ml of nitrofurantoin.

FIG. 11 illustrates an embodiment of a method 1100 for detecting asusceptibility of an infectious agent 102 to one or more anti-infectives104. The method 1100 can include exposing a surface, such as the filtersurface 126 or the substrate surface 202, comprising the infectiousagent 102 with a first solution, such as the nutrient solution 130, in astep 1102. The method 1100 can also include separating the firstsolution from the infectious agent 102 after exposing the surface in astep 1104. The method 1100 can further include monitoring an electricalcharacteristic 800 of a sensor 116 upon introducing the first solutionto the sensor 116 in a step 1106. The method 1100 can also includeexposing the surface comprising the infectious agent 102 with a secondsolution, such as additional nutrient solution 130, wherein the secondsolution comprises an anti-infective 104 in a step 1108. The method 1100can further include separating the second solution from the infectiousagent 102 after exposing the surface in a step 1110. The method 1100 canalso include detecting any changes in the electrical characteristic 800of the sensor 116 after introducing the second solution to the sensor116 in a step 1112. The method 1100 can further include assessing thesusceptibility of the infectious agent 102 to the anti-infective 104using any detected changes in the electrical characteristic 800 of thesensor 116 in a step 1114.

FIG. 12 illustrates another method 1200 for detecting a susceptibilityof an infectious agent 102 to one or more anti-infectives 104. Themethod 1200 can include exposing a surface, such as the filter surface126 or the substrate surface 202, comprising the infectious agent 102with a first solution, such as the nutrient solution 130, in a step1202. The method 1200 can also include separating the first solutionfrom the infectious agent 102 after exposing the surface in a step 1204.The method 1200 can further include monitoring a first electricalcharacteristic of a first sensor, such as the control sensor 122, uponintroducing the first solution to the first sensor in a step 1206. Themethod 1200 can also include exposing the surface comprising theinfectious agent 102 with a second solution, such as additional nutrientsolution 130, wherein the second solution comprises an anti-infective104 in a step 1208. The method 1200 can further include separating thesecond solution from the infectious agent 102 after exposing the surfacein a step 1210. The method 1200 can also include monitoring a secondelectrical characteristic of a second sensor, such as the active sensor120, after introducing the second solution to the second sensor in astep 1212. The method 1200 can further include comparing the firstelectrical characteristic and the second electrical characteristic toassess the susceptibility of the infectious agent 102 to theanti-infective 104 in a step 1214.

FIG. 13 illustrates another method 1300 for detecting a susceptibilityof an infectious agent 102 to one or more anti-infectives 104. Themethod 1300 can include exposing a filter 110 comprising an infectiousagent 102 to a first solution, such as the nutrient solution 130 in astep 1302. The method 1300 can also include incubating the filter 110comprising the infectious agent 102 and the first solution in a step1304. The method 1300 can also include separating the first solutionfrom the infectious agent 102 after incubating the filter 110 in a step1306. The method 1300 can also include monitoring a first solutioncharacteristic of the first solution using a sensor or a sensor device300, such as an ISFET sensor, in a step 1308. The method 1300 can alsoinclude exposing the filter 110 comprising the infectious agent 102 to asecond solution, wherein the second solution comprises an anti-infective104 in a step 1310. The method 1300 can also include incubating thefilter 110 comprising the infectious agent 102 and the second solutionin a step 1312. The method 1300 can also include separating the secondsolution from the infectious agent 102 after incubating the filter 110in a step 1314. The method 1300 can also include monitoring a secondsolution characteristic of the second solution using the sensor 116 in astep 1316. The method 1300 can also include comparing the first solutioncharacteristic and the second solution characteristic to assess thesusceptibility of the infectious agent 102 to the anti-infective 104 ina step 1318.

FIG. 14 illustrates another method 1400 for detecting a susceptibilityof an infectious agent 102 to one or more anti-infectives 104. Themethod 1400 can include temporarily exposing a solution, such as thenutrient solution 130, to the infectious agent 102, wherein the solutioncomprises an anti-infective 104 in a step 1402. The method 1400 can alsoinclude providing a sensor 116 in fluid communication with the solutionafter the solution is separated from the infectious agent 102, whereinthe sensor 116 comprises an electrical characteristic 800 in a step1404. The method 1400 can also include monitoring the sensor 116 for achange to the electrical characteristic 800 of the sensor 116 afterproviding the sensor 116 in fluid communication with the solution in astep 1406. The method 1400 can also include providing an indication ofthe susceptibility of the infectious agent 102 to the anti-infective 104upon a failure to detect the change to the electrical characteristic 800of the sensor 116 in a step 1408.

FIG. 15 illustrates another method 1500 for detecting a susceptibilityof an infectious agent 102 to one or more anti-infectives 104. Themethod 1500 can include delivering a first solution to a surface, suchas the filter surface 126 or the substrate surface 202, wherein thefirst solution does not contain an anti-infective 104 and wherein aninfectious agent 102 is located on the surface in a step 1502. Themethod 1500 can also include separating the first solution from theinfectious agent 102 and the surface in a step 1504. The method 1500 canfurther include fluidly coupling a sensor 116 with the first solution ina step 1506. The method 1500 can also include monitoring an electricalcharacteristic 800 of the sensor 116 while the sensor 116 is fluidlycoupled to the first solution in a step 1508. The method 1500 canfurther include delivering a second solution to the surface, where thesecond solution comprises the anti-infective 104 in a step 1510. Themethod 1500 can also include separating the second solution from theinfectious agent 102 and the surface in a step 1512. The method 1500 canfurther include monitoring the sensor 116 for a change in the electricalcharacteristic 800 while the sensor 116 is fluidly coupled to the secondsolution to assess the susceptibility of the infectious agent 102 to theanti-infective 104 in a step 1514.

FIG. 16 illustrates another method 1600 for detecting a susceptibilityof an infectious agent 102 to one or more anti-infectives 104. Themethod 1600 can include introducing a fluid sample 124 to a firstsurface, such as the first filter surface 126A, and a second surface,such as the second filter surface 126B, in a step 1602. The method 1600can also include exposing the first surface to a first solution, such asthe nutrient solution 130, in a step 1604. The first surface cancomprise the infectious agent 102 when the infectious agent 102 ispresent in the fluid sample 124.

The method 1600 can also include exposing the second surface to a secondsolution, such as additional instances of the nutrient solution 130 in astep 1606. The second surface can comprise one or more anti-infectives104 or anti-infectives of differing concentrations. The second surfacecan also comprise the infectious agent 102 when the infectious agent 102is present in the fluid sample 124.

The method 1600 can also include sampling the first solution afterexposing the first solution to the first surface in step 1608. Samplingthe first solution can include sampling the effluent or outflow of thefirst solution, such as the first sample effluent 134A. In oneembodiment, sampling the first solution can also involve separating thefirst solution from the first surface so the first solution is not influid communication with the first surface, the infectious agent 102 onthe first surface, or a combination thereof when sampled. The method1600 can also include sampling the second solution after exposing thesecond solution to the second surface in step 1610. Sampling the secondsolution can include sampling the effluent or outflow of the secondsolution, such as the second sample effluent 134B. In one embodiment,sampling the second solution can also involve separating the secondsolution from the second surface so the second solution is not in fluidcommunication with the second surface, the infectious agent 102 on thesecond surface, or a combination thereof when sampled.

The method 1600 can also include monitoring a first electricalcharacteristic of a first sensor 116A exposed to the first solutionsampled in step 1612. The method 1600 can also include monitoring asecond electrical characteristic of a second sensor 116B exposed to thesecond solution sampled in step 1614. The method 1600 can furtherinclude comparing the first electrical characteristic and the secondelectrical characteristic to assess the susceptibility of the infectiousagent 102 to the anti-infective 104 in step 1616.

FIG. 17 illustrates another embodiment of the system 100 for detectingor assessing the susceptibility of an infectious agent 102 to ananti-infective 104. The infectious agent 102 can be a bacteria, afungus, a virus, or a prion.

The system 100 can comprise the fluid delivery device 106, a firstfilter housing 108A containing a first filter 110A, a second filterhousing 108B containing a second filter 110B, a first sensor 116A, asecond sensor 116B, and the reader 114. The first sensor 116A can be anyof the control sensor 122 or the active sensor 120. The second sensor116B can also be any of the active sensor 120 or the control sensor 122.

In an alternative embodiment contemplated by the present disclosure butnot shown in FIG. 17, the system 100 can comprise the fluid deliverydevice 106, a first filter housing 108A containing a first filter 110A,a second filter housing 108B containing a second filter 110B, a sensor116, and the reader 114.

In some instances, the fluid sample 124 can contain the infectious agent102. The system 100 can detect or assess the level of susceptibility ofthe infectious agent 102 in the fluid sample 124 to an anti-infective104. The system 100 can also be used to initially determine the presenceor absence of an infectious agent 102 in the fluid sample 124.

As illustrated in FIG. 17, the fluid delivery device 106 can deliver orinject the fluid sample 124 into the first filter housing 108A and thesecond filter housing 108B in step 17A. The fluid delivery device 106can be a pump. For example, the fluid delivery device 106 can be ahydraulic pump, a pneumatic pump, a syringe pump, or a combinationthereof. In other embodiments, the fluid delivery device 106 can be aninjection cartridge, a microfluidic channel, a pipette, a reaction tube,a capillary, a test tube, a combination thereof, or a portion therein.

The first filter housing 108A or the second filter housing 108B can be acontainer or vessel configured to secure or enclose the first filter110A or the second filter 110B, respectively. For example, the firstfilter housing 108A or the second filter housing 108B can be aprotective chamber. The protective chamber can be an electricallyisolated environment. The protective chamber can also be a temperaturecontrolled chamber, a light controlled chamber, or a combinationthereof.

The first filter 110A, the second filter 110B, or a combination thereofcan be a non-clogging filter. The first filter surface 126A can be anon-clogging filter surface. The second filter surface 126B can also bea non-clogging filter surface. The first filter 110A, the second filter110B, or a combination thereof can also have filter pores ofsequentially smaller pore size. For example, the first filter 110A, thesecond filter 110B, or a combination thereof can have larger filterpores at the top of the filter and progressively smaller filters porestoward the bottom of the filter. Although not shown in FIG. 17, it iscontemplated by this disclosure that the first filter 110A or the secondfilter 110B can refer to a plurality of filters in a stackedarrangement.

The first filter 110A can comprise the infectious agent 102 when thefluid sample 124 introduced to the first filter 110A comprises orcarries the infectious agent 102. The second filter 110B can alsocomprise the infectious agent 102 when the fluid sample 124 introducedto the second filter 110B comprises or carries the infectious agent 102.

The first filter 110A can be a mesh or matrix structure for isolating orseparating the infectious agent 102 or other molecules or cells from thesupernatant of the fluid sample 124. The second filter 110B can also bea mesh or matrix structure for isolating or separating the infectiousagent 102 or other molecules or cells from the supernatant of the fluidsample 124. In certain embodiments, the first filter 110A or the secondfilter 110B can be selected from the group consisting of celluloseacetate, regenerated cellulose, nylon, polystyrene, polyvinylidenefluoride (PVDF), polyethersulfone (PES), polytetrafluorethylene (PTFE),or a combination thereof.

The first filter 110A can comprise a first filter surface 126A. Thefirst filter surface 126A can be the portion of the first filter 110Aused to isolate or trap the infectious agent 102. The first filtersurface 126A can include an external surface, an internal surfaceextending into the first filter 110A, or a combination thereof.

The second filter 110B can comprise a second filter surface 126B. Thesecond filter surface 126B can be the portion of the second filter 110Bused to isolate or trap the infectious agent 102. The second filtersurface 126B can include an external surface, an internal surfaceextending into the second filter 110B, or a combination thereof.

The second filter 110B or the second filter surface 126B can comprisethe anti-infective 104. The anti-infective 104 can be added orintroduced to the second filter surface 126B before or after exposingthe second filter surface 126B to the fluid sample 124.

In another embodiment, the anti-infective 104 can be incorporated orembedded into or coated onto the second filter 108B or the second filtersurface 126B before exposing the second filter 110B or the second filtersurface 126B to the fluid sample 124.

In yet another embodiment, the anti-infective 104 can be introducedthrough a solution exposed to the first filter 110A, the second filter110B, or a combination thereof. For example, the anti-infective 104 canbe introduced through the nutrient solution 130.

The anti-infective 104 can comprise a bacteriostatic anti-infective, abactericidal anti-infective, an anti-fungal anti-infective, an antiviralanti-infective, a prion inhibitor, or a combination thereof.

In another embodiment, the anti-infective 104 can be a bacterial growthinhibitor or stimulator. The bacterial growth inhibitor or stimulatorcan selectively inhibit or promote the growth of gram positive or gramnegative bacteria. The bacterial growth inhibitor or stimulator cancomprise a dye or a chemical compound. In some embodiments, the dye caninclude, but is not limited to, Methylene blue, Bromothymol blue, EosinB, Safranin O, Crystal violet, or a combination thereof. The chemicalcompound can include, but is not limited to, sodium azide, bile acids,high sodium chloride, or a combination thereof. The anti-infective 104can also comprise a carbon source other than glucose, such as lactose ormannose, to select for certain bacterial species. The bacterial growthinhibitor, the carbon source, or a combination thereof can also be addedto the nutrient solution 130

The first filter housing 108A or the second filter housing 108B can haveat least one opening which allows fluid or supernatant from the fluidsample 124 to evacuate the first filter housing 108A or the secondfilter housing 108B. For example, step 17A can include the additionalstep of discarding the fluid or supernatant from the fluid sample 124through the opening after isolating the infectious agent 102 on thefirst filter surface 126A or the second filter surface 126B.

In an alternative embodiment not shown in FIG. 17, a stimulus solutioncan be added to the fluid sample 124 before introducing the fluid sample124 to the first filter 110A or the second filter 110B. The stimulussolution can be a nutrient or growth solution. The stimulus solution canhave a different composition than nutrient solution 130. The stimulussolution can be a super nutrient solution.

The fluid sample 124 can also be pre-filtered in a step before step 17A.This pre-filtering step can involve filtering the fluid sample 124 usinga filter, a microfluidic filter, or a combination thereof to filter outother larger cellular components including blood cells or epithelialcells from the fluid sample 124 when the fluid sample 124 is composed ofbodily fluid.

The same fluid delivery device 106 or another fluid delivery device 106can also be used to deliver or inject nutrient solution 130 to the firstfilter housing 108A, the second filter housing 108B, or a combinationthereof in step 17B. The fluid delivery device 106 can continuously orperiodically expose the first filter surface 126A, the second filtersurface 126B, or a combination thereof to the nutrient solution 130.

After exposing the first filter 110A or the second filter 110B to thenutrient solution 130, the first filter 110A or the second filter 110Bcan be heated to a temperature of between 30° C. and 40° C. and allowedto incubate for an incubation period 132 in step 17C. In one embodiment,the first filter 110A or the second filter 110B can be incubated whilein the first filter housing 108A or the second filter housing 108B,respectively. In another embodiment, the first filter 110A or the secondfilter 110B can be removed from the first filter housing 108A or thesecond filter housing 108B, respectively, prior to incubation. In someembodiments, the first filter 110A, the second filter 110B, or acombination thereof can be incubated with the nutrient solution 130. Theincubation period 132 can range from 15 minutes to over one hour. Inother embodiments, the incubation period 132 can be less than 15minutes. The incubation period 132 can be adjusted based on the type ofinfectious agent 102, such as the type of bacteria, fungus, virus, orprion.

The incubation period 132 can also be adjusted based on the amount ofthe infectious agent 102 present in the fluid sample 124. For example,the incubation period 132 can be increased when the amount of theinfectious agent 102 is below a threshold amount. The first filter 110Aor the second filter 110B can be allowed to incubate with the nutrientsolution 130 in order to promote the proliferation of the infectiousagent 102 on the first filter surface 126A or the second filter surface126B, respectively. One advantage of incubating the first filter 110Aand the second filter 110B is to increase the sensitivity of the system100 to small amounts of the infectious agent 102. For example,incubating the first filter 110A and the second filter 110B can allowthe system 100 to reduce its level of detection.

After incubating the first filter 110A or the second filter 110B, theeffluent or outflow of the nutrient solution 130 exposed to the firstfilter 110A or the second filter 110B can be sampled. The effluent oroutflow of the nutrient solution 130 exposed to the first filter 110Acan be referred to as the first sample effluent 134A. The first sampleeffluent 134A can be sampled by a first sensor 116A in step 17D. Thefirst sample effluent 134A can be sampled by applying or introducing analiquot of the first sample effluent 134A to the first sensor 116A. Inanother embodiment, the first sample effluent 134A can be sampled byinserting a portion of the first sensor 116A directly into the firstsample effluent 134A.

The effluent or outflow of the nutrient solution 130 exposed to thesecond filter 110B can be referred to as the second sample effluent134B. The second sample effluent 134B can be sampled by a second sensor116B in step 17E. The second sample effluent 134B can be sampled byapplying or introducing an aliquot of the second sample effluent 134B tothe second sensor 116B. In another embodiment, the second sampleeffluent 134B can be sampled by inserting a portion of the second sensor116B directly into the second sample effluent 134B.

The first sample effluent 134A and the second sample effluent 134B caneach comprise a solution characteristic 136. The solution characteristic136 can refer to one or more attributes of the solution making up thefirst sample effluent 134A, the second sample effluent 134B, or acombination thereof. For example, the solution characteristic 136 caninclude a concentration of a solute, an absolute number or molecularcount of solutes in solution, a solution temperature, or a combinationthereof. For example, the solution characteristic 136 can refer to theamount or concentration of ions, organic molecules such as amino acids,minerals, or other inorganic compounds in the sample effluent 134.

The solution characteristic 136 can vary as a result of natural changesdue to the energy use, growth, and metabolism of the infectious agent102. For example, the solution characteristic 136 can be a direct orindirect byproduct of a cellular activity undertaken by the infectiousagent 102 such as cell metabolism or cell growth. The solutioncharacteristic 136 can vary as a result of ions, organic molecules, orminerals produced by or attributed to the infectious agent 102 on thefirst filter surface 126A, the second filter surface 126B, or acombination thereof.

In one embodiment, the first sample effluent 134A, the second sampleeffluent 134B, or a combination thereof can comprise hydrogen ions (H⁺)as a byproduct of bacterial cell metabolism or growth. In otherembodiments, the first sample effluent 134A, the second sample effluent134B, or a combination thereof can comprise adenosine triphosphate(ATP), carbon dioxide (CO₂), lactic acid, carbonic acid, nitrates (NO₃⁻), or a combination thereof produced by or attributed to the infectiousagent 102.

In an alternative embodiment contemplated by the present disclosure, thesame sensor 116 can be used to sample the first sample effluent 134A andthe second sample effluent 134B.

In yet another embodiment, the first sensor 116A, the second sensor116B, or the one sensor 116, can be integrated into the first filter110A, the second filter 110B, or a combination thereof. For example, thefirst sensor 116A can be integrated into the first filter 110A and thesecond sensor 116B can be integrated into the second filter 110B.

The reader 114 can monitor an electrical characteristic 800 (see FIG. 8)of the first sensor 116A exposed to the first sample effluent 134A instep 17F. The reader 114 can also monitor the electrical characteristic800 of the second sensor 116B exposed to the second sample effluent 134Bin step 17F. In this embodiment, the electrical characteristic 800 ofthe first sensor 116A can be referred to as a first electricalcharacteristic and the electrical characteristic 800 of the secondsensor 116B can be referred to as the second electrical characteristic.

When only one sensor 116 is used to sample the sample effluents, thereader 114 can monitor the electrical characteristic 800 of the onesensor 116 exposed to the first sample effluent 134A and the reader 114can also monitor the electrical characteristic 800 of the one sensor 116exposed to the second sample effluent 134B. In this embodiment, theelectrical characteristic 800 of the sensor 116 while sampling the firstsample effluent 134A can be referred to as the first electricalcharacteristic and the electrical characteristic 800 of the sensor 116while sampling the second sample effluent 134B can be referred to as thesecond electrical characteristic.

The electrical characteristic 800 can include a current, a voltage, athreshold voltage, a capacitance, a resistance, a noise level, asubthreshold swing, a level of induction, or a combination thereofmeasured at or near the sensor 116. The reader 114 can be electricallyor communicatively coupled to the first sensor 116A, the second sensor116B, or a combination thereof to monitor the electrical characteristic800 of the first sensor 116A, the second sensor 116B, or a combinationthereof over time. The reader 114 can also be configured to provide aread-out of the electrical characteristic 800 of the first sensor 116A,the second sensor 116B, or a combination thereof. When only one sensor116 is used to sample the sample effluents, the reader 114 can beelectrically or communicatively coupled to the one sensor 116.

In certain embodiments, the reader 114 can be a mobile device, ahandheld device, a tablet device, or a computing device such as a laptopor desktop computer. The reader 114 can compare the first electricalcharacteristic with the second electrical characteristic to assess thesusceptibility of the infectious agent 102 to the anti-infective 104.

The first electrical characteristic can differ from the secondelectrical characteristic when the solution characteristic 136 of thefirst sample effluent 134A differs from the solution characteristic 136of the second sample effluent 134B as a result of differences in thesolution temperature, the concentration of solutes present in the sampleeffluents, or the amount of solutes present in the sample effluents. Forexample, the first electrical characteristic and the second electricalcharacteristic can differ when the solution characteristic 136 of thefirst sample effluent 134A and the solution characteristic of the secondsample effluent 134B differ in their pH, temperature, the concentrationof another ion, or a combination thereof.

The reader 114 can assess the susceptibility of the infectious agent 102to the anti-infective 104 as a binary assessment or a gradated or tieredassessment. In one embodiment, the reader 114 can assess thesusceptibility of the infectious agent 102 as either resistant ornon-resistant to the anti-infective 104. In this embodiment, the secondfilter 110B or the second filter surface 126B can comprise a set amountof the anti-infective 104. The reader 114 can then assess thesusceptibility of the infectious agent 102 as either resistant ornon-resistant based on any detected differences in first electricalcharacteristic and the second electrical characteristic.

The reader 114 can assess the susceptibility of the infectious agent 102as not resistant to the anti-infective 104 when the reader 114 fails todetect a difference or a statistically significant difference betweenthe first electrical characteristic and the second electricalcharacteristic. More specifically, a statistically significantdifference in the electrical characteristic can be a differenceexceeding a threshold value.

In other embodiments, the reader 114 can assess the level ofsusceptibility of the infectious agent 102 on a gradated or tieredscale. For example, the reader 114 can assess the susceptibility of theinfectious agent 102 as being resistant, mildly susceptible, orsusceptible to the anti-infective 104. In these embodiments, additionalfilter surfaces, including the second filter surface 126B and a thirdfilter surface, can be used which comprise anti-infectives 104 ofdifferent concentrations. While three categories of susceptibility arediscussed, it should be understood by one of ordinary skill in the artthat four or greater categories of susceptibility or four or greaterfilters can be used to assess the level of susceptibility of theinfectious agent 102 to differing concentrations of the anti-infective104.

FIG. 18 illustrates another method 1600 for detecting a susceptibilityof an infectious agent 102 to one or more anti-infectives 104. Themethod 1600 can include introducing a fluid sample 124 to a firstsurface, such as the first filter surface 126A, and a second surface,such as the second filter surface 126B, in a step 1802. The method 1800can also include exposing the first surface to a first solution, such asthe nutrient solution 130, in a step 1804. The first surface cancomprise the infectious agent 102 when the infectious agent 102 ispresent in the fluid sample 124.

The method 1800 can also include exposing the second surface to a secondsolution, such as additional instances of the nutrient solution 130 in astep 1806. The second surface can comprise one or more anti-infectives104 or anti-infectives of differing concentrations. The second surfacecan also comprise the infectious agent 102 when the infectious agent 102is present in the fluid sample 124.

The method 1800 can also include sampling the first solution afterexposing the first solution to the first surface in step 1808. Samplingthe first solution can include sampling the effluent or outflow of thefirst solution, such as the first sample effluent 134A. In oneembodiment, sampling the first solution can also involve separating thefirst solution from the first surface so the first solution is not influid communication with the first surface, the infectious agent 102 onthe first surface, or a combination thereof. The method 1800 can alsoinclude sampling the second solution after exposing the second solutionto the second surface in step 1810. Sampling the second solution caninclude sampling the effluent or outflow of the second solution, such asthe second sample effluent 134B. In one embodiment, sampling the secondsolution can also involve separating the second solution from the secondsurface so the second solution is not in fluid communication with thesecond surface, the infectious agent 102 on the second surface, or acombination thereof.

The method 1800 can also include monitoring a first electricalcharacteristic of a sensor 116 exposed to the first solution sampled instep 1812. The method 1800 can also include monitoring a secondelectrical characteristic of the sensor 116 exposed to the secondsolution sampled in step 1814. The method 1800 can further includecomparing the first electrical characteristic and the second electricalcharacteristic to assess the susceptibility of the infectious agent 102to the anti-infective 104 in step 1816.

Each of the individual variations or embodiments described andillustrated herein has discrete components and features which may bereadily separated from or combined with the features of any of the othervariations or embodiments. Modifications may be made to adapt aparticular situation, material, composition of matter, process, processact(s) or step(s) to the objective(s), spirit or scope of the presentinvention.

Methods recited herein may be carried out in any order of the recitedevents that is logically possible, as well as the recited order ofevents. For example, the flowcharts or process flows depicted in thefigures do not require the particular order shown to achieve the desiredresult. Moreover, additional steps or operations may be provided orsteps or operations may be eliminated to achieve the desired result.

It will be understood by one of ordinary skill in the art that all or aportion of the methods disclosed herein may be embodied in anon-transitory machine readable or accessible medium comprisinginstructions readable or executable by a processor or processing unit ofa computing device or other type of machine.

Furthermore, where a range of values is provided, every interveningvalue between the upper and lower limit of that range and any otherstated or intervening value in that stated range is encompassed withinthe invention. Also, any optional feature of the inventive variationsdescribed may be set forth and claimed independently, or in combinationwith any one or more of the features described herein.

All existing subject matter mentioned herein (e.g., publications,patents, patent applications and hardware) is incorporated by referenceherein in its entirety except insofar as the subject matter may conflictwith that of the present invention (in which case what is present hereinshall prevail). The referenced items are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such material by virtue of prior invention.

Reference to a singular item, includes the possibility that there areplural of the same items present. More specifically, as used herein andin the appended claims, the singular forms “a,” “an,” “said” and “the”include plural referents unless the context clearly dictates otherwise.It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

This disclosure is not intended to be limited to the scope of theparticular forms set forth, but is intended to cover alternatives,modifications, and equivalents of the variations or embodimentsdescribed herein. Further, the scope of the disclosure fully encompassesother variations or embodiments that may become obvious to those skilledin the art in view of this disclosure. The scope of the presentinvention is limited only by the appended claims.

1.-20. (canceled)
 21. A method for detecting a susceptibility of amicroorganism to an antibiotic, the method comprising: exposing asurface comprising the microorganism to a first solution; separating thefirst solution from the microorganism after the first solution isexposed to the surface; monitoring an electrical characteristic of asensor upon introducing the first solution to the sensor; exposing thesurface comprising the microorganism to a second solution, wherein thesecond solution comprises an antibiotic; separating the second solutionfrom the microorganism after the second solution is exposed to thesurface; detecting any changes in the electrical characteristic of thesensor after introducing the second solution to the sensor; andassessing the susceptibility of the microorganism to the antibioticusing any detected changes in the electrical characteristic.
 22. Themethod of claim 21, wherein the surface is at least one of a filtersurface and a well surface.
 23. The method of claim 21, wherein thechange in the electrical characteristic of the sensor indicates a changein a solution characteristic.
 24. The method of claim 23, wherein thechange in the solution characteristic is a change in a concentration ofan ion.
 25. The method of claim 21, wherein the susceptibility of themicroorganism to the antibiotic is assessed between 60 minutes to 240minutes.
 26. The method of claim 21, wherein the sensor comprises a gatedielectric layer selected from a group consisting of an aluminum oxidelayer, a hafnium oxide layer, a zirconium oxide layer, a hafniumsilicate layer, a zirconium silicate layer, or any combination thereof.27. The method of claim 21, wherein the sensor is an ion-sensitive fieldeffect transistor.
 28. The method of claim 21, wherein the microorganismcomprises bacteria selected from the genera consisting of Acinetobacter,Aeromonas, Bacillus, Bacteroides, Citrobacter, Enterobacter,Escherichia, Klebsiella, Morganella, Pandoraea, Proteus, Providencia,Pseudomonas, Ralstonia, Raoultella, Salmonella, Serratia, Shewanella,Shigella, Stenotrophomonas, Streptomyces, or any combination thereof.29. The method of claim 21, wherein the antibiotic comprises abacteriostatic antibiotic selected from the group consisting ofβ-lactams, Aminoglycosides, Ansamycins Glycopeptides, Lipopeptides,Quinolones, Streptogramins, or any combination thereof.
 30. A method fordetecting a susceptibility of a microorganism to an antibiotic, themethod comprising: exposing a surface comprising the microorganism to afirst solution; separating the first solution from the microorganismafter the first solution is exposed to the surface; monitoring a firstelectrical characteristic of a first sensor upon introducing the firstsolution to the first sensor; exposing the surface comprising themicroorganism to a second solution, wherein the second solutioncomprises an antibiotic; separating the second solution from themicroorganism after the second solution is exposed to the surface;monitoring a second electrical characteristic of a second sensor afterintroducing the second solution to the second sensor; and comparing thefirst electrical characteristic and the second electrical characteristicto assess the susceptibility of the microorganism to the antibiotic. 31.The method of claim 30, wherein the surface is at least one of a filtersurface and a well surface.
 32. The method of claim 30, whereincomparing the first electrical characteristic and the second electricalcharacteristic includes determining a difference between the firstelectrical characteristic and the second electrical characteristic andthe difference between the first electrical characteristic and thesecond electrical characteristic is a result of a difference in asolution characteristic of the first solution and the second solution.33. The method of claim 32, wherein the difference in the solutioncharacteristic of the first solution and the second solution is adifference in a concentration of an ion between the first solution andthe second solution.
 34. The method of claim 30, wherein thesusceptibility of the microorganism to the antibiotic is assessedbetween 60 minutes to 240 minutes.
 35. The method of claim 30, whereinat least one of the first sensor and the second sensor comprises a gatedielectric layer selected from a group consisting of an aluminum oxidelayer, a hafnium oxide layer, a zirconium oxide layer, a hafniumsilicate layer, a zirconium silicate layer, or any combination thereof.36. The method of claim 30, wherein at least one of the first sensor andthe second sensor is an ion-sensitive field effect transistor.
 37. Themethod of claim 30, wherein the microorganism comprises bacteriaselected from the genera consisting of Acinetobacter, Aeromonas,Bacillus, Bacteroides, Citrobacter, Enterobacter, Escherichia,Klebsiella, Morganella, Pandoraea, Proteus, Providencia, Pseudomonas,Ralstonia, Raoultella, Salmonella, Serratia, Shewanella, Shigella,Stenotrophomonas, Streptomyces, or any combination thereof.
 38. Themethod of claim 30, wherein the antibiotic comprises a bacteriostaticantibiotic selected from the group consisting of β-lactams,Aminoglycosides, Ansamycins Glycopeptides, Lipopeptides, Quinolones,Streptogramins, or any combination thereof.
 39. A method for detecting asusceptibility of a microorganism to an antibiotic, the methodcomprising: temporarily exposing a solution to the microorganism,wherein the solution comprises an antibiotic; providing a sensor influid communication with the solution after the solution temporarilyexposed to the microorganism is separated from the microorganism,wherein the sensor comprises an electrical characteristic; monitoringthe sensor for a change in the electrical characteristic of the sensorafter providing the sensor in fluid communication with the solution;providing an indication of the susceptibility of the microorganism tothe antibiotic upon a failure to detect the change in the electricalcharacteristic of the sensor.
 40. The method of claim 39, wherein thechange in the electrical characteristic of the sensor indicates a changein a concentration of an ion in the solution.